Screening methods for compounds useful in the regulation of body weight

ABSTRACT

The present invention relates to drug screening assays, and diagnostic and therapeutic methods for the treatment of body weight disorders, such as obesity, anorexia and cachexia, utilizing the melanocortin 4-receptor (MC4-R) as the target for intervention. The invention also relates to compounds that modulate the activity or expression of the MC4-R, and the use of such compounds in the treatment of body weight disorders.

1. INTRODUCTION

[0001] The present invention relates to drug screening assays, anddiagnostic and therapeutic methods for the treatment of body weightdisorders, such as obesity, anorexia and cachexia, involving themelanocortin 4-receptor (MC4-R). The invention also relates to compoundsthat modulate the activity or expression of the MC4-R, and the use ofsuch compounds in the treatment of body weight disorders.

2. BACKGROUND OF THE INVENTION

[0002] Melanocortins (a variety of different peptide products resultingfrom post-translational processing of pro-opiomelanocortin) are known tohave a broad array of physiological actions. Aside from their well knowneffects on adrenal cortical function (e.g., by ACTH, adrenocorticotropichormone), and on melanocytes (e.g., by α-MSH, melanocyte stimulatinghormone), melanocortins have been shown to affect behavior, learning,and memory, control of the cardiovascular system, analgesia,thermoregulation, and the release of other neurohumoral agents includingprolactin, luteinizing hormone, and biogenic amines. Peripherally,melanocortins have been identified to have immunomodulatory andneurotrophic properties and to be involved in events surroundingparturition.

[0003] The melanocortins mediate their effects through melanocortinreceptors (MC-R)—a subfamily of G-protein coupled receptors. Other thanthe MC1-R which was identified as specific for α-MSH, and MC2-R whichwas identified as specific for ACTH, the melanocortin receptors clonedand identified to date (MC3-R, MC4-R, MC5-R) are thought of as “orphan”receptors—i.e., the identity of the native ligand for each receptorremains unidentified, and the physiologic function of each receptor typeremains unknown.

[0004] The agouti protein is a gene product expressed in mice that isknown to be involved in determining coat color, but also thought to playa role in obesity when its normal expression pattern is de-regulated andthe protein is ubiquitously expressed. The receptor for agouti has notbeen identified or cloned; however, it has been observed that agoutiantagonizes the MSH-induced activation of two melanocortin receptors.

[0005] 2.1. The Melanocortin Receptors

[0006] The first two melanocortin receptors cloned were the melanocyteMSH receptor, MC1-R, and the adrenocortical ACTH receptor, MC2-R(Mountjoy et al., 1992, Science 257:1248-1251; Chhajlani & Wikberg,1992, FEBS Lett. 309:417-420). Subsequently, three additionalmelanocortin receptor genes were cloned which recognize the coreheptapeptide sequence (MEHFRWG) of melanocortins. Two of these receptorshave been shown to be expressed primarily in the brain, MC3-R(Roselli-Rehfuss et al., 1993, Proc. Natl. Acad. Sci. USA 90:8856-8860;Gantz et al., 1993, J. Biol. Chem. 268:8246-8250) and MC4-R (Gantz etal., 1993, J. Biol. Chem. 268:15174-15179; Mountjoy et al., 1994, Mol.Endo. 8:1298-1308). A fifth nelanocortin receptor (originally calledMC2-R) is expressed in numerous peripheral organs as well as the brain(Chhajlani et al., 1993, Biochem. Biophys. Res. Commun. 195:866-873;Gantz et al., 1994, Biochem. Biophs. Res. Commun. 200:1214-1220). Thenative ligands and functions of these latter three receptors remainunknown.

[0007] Because of their “orphan” status as receptors without anidentified ligand, and the absence of any known physiological role forthese new receptors, investigators have attempted to characterize thereceptors in vitro, by their ability to bind and respond (e.g.,transduce signal) to a variety of known melanocortins (e.g., seeRoselli-Rehfuss, 1993, supra; and Gantz, 1993 supra) or agonists andantagonists derived from MSH and ACTH amino acid sequences (e.g., seeHruby et al., 1995, J. Med. Chem. 38:3454-3461; and Adan et al., 1994,Eur. J. Pharmacol. 269:331-337). In another approach, the members of themelanocortin receptor family were differentiated on the basis of theirpattern of tissue distribution as a means for hypothesizing a function(e.g., See Gantz, 1993, supra; and Mountjoy 1994, supra). For example,expression of MC1-R is localized to melanocytes, MC2-R is localized toadrenal cortical cells, whereas the MC3-R and MC4-R are found primarilyin the brain but not in the adrenal cortex or melanocytes; MC4-R is notexpressed in the placenta, a tissue that expresses large amounts ofMC3-R. Based upon its expression pattern in the hippocampal region ofthe brain, a role for the MC4-R in learning and memory was proposed(Gantz, 1993, supra) but was noted to be a “pharmacological paradox” inthat the MC4-R does not respond well to compounds known to have aneffect on retention of learned behaviors. (Mountjoy, 1994, supra).Mountjoy 1994 further suggests that the MC4-R may participate inmodulating the flow of visual and sensory information, or coordinateaspects of somatomotor control, and/or may participate in the modulationof autonomic outflow to the heart.

[0008] Thus, despite such efforts, the native ligands and function ofMC3-R, MC4-R and MC5-R remain elusive.

[0009] 2.2. The Agouti Protein

[0010] The agouti gene is predicted to encode a secreted proteinexpressed in hair follicles and the epidermis, the expression of whichcorrelates with the synthesis of the yellow pigment associated with theagouti phenotype (Miller et al., 1993, Gene & Development 7:454-467).Certain dominant mutations of the agouti gene result in de-regulated,ubiquitous expression of the agouti protein in mice, demonstratingpleiotropic effects that include obesity and increased tumorsusceptibility. (Miller et al., 1993, supra; Michaud et al., 1993, Genes& Development 7:1203-1213). Ectopic expression of the normal, wild-type,agouti protein in transgenic mice results in obesity, diabetes, and theyellow coat color commonly observed in spontaneous obese mutants(Klebig, et al., 1995, Proc. Natl. Acad. Sci. USA 92:4728-4732). Forreviews, see Jackson, 1993, Nature 362:587-588; Conklin & Bourne, 1993,Nature 364:110; Siracusa 1994, TIG 10:423-428; Yen et al., 1994, FASEBJ. 8:479-488; Ezzell, 1994, J. NIH Res. 6:31-33; and Manne et al., 1995,Proc. Sci.-USA 92:4721-4724.

[0011] No receptor for agouti has been identified. Agouti has beenreported to be a competitive antagonist of αMSH binding to the MC1-R andMC4-R in vitro (Lu et al., 1996, Nature 371:799-802), and the authorsspeculated that ectopic expression of agouti may lead to obesity byantagonism of melanocortin receptors expressed outside the hairfollicle. In this regard, a number of theories have been proposed toaccount for the induction of obesity by ectopic expression of agouti.For example, agouti expression in skeletal muscle may result in insulinresistance, hyperinsulinemia and obesity via elevation of Ca²+levels;alternatively ectopic agouti expression in adipocytes may depresslipolysis; conversely direct effects of agouti on pancreatic β isletcells may result in hyperinsulinemia and obesity; yet anotherpossibility is that agouti expression in the brain may result in obesitydue to a primary effect on areas of the brain controlling weightregulation and insulin production (see Klebig 1995, supra).

[0012] In sum, the mechanism of agouti-induced obesity in mice isunknown, and the relevance, if any, of this phenomenon to human obesephenotypes has not been established.

3. SUMMARY OF THE INVENTION

[0013] The present invention relates to drug screening assays toidentify compounds for the treatment of body weight disorders, such asobesity, anorexia and cachexia by using MC4-R as a target. The inventionalso relates to compounds that modulate body weight via the MC4-R. Thepresent invention also relates to the treatment of body weight disordersby targeting the MC4-R.

[0014] The invention is based, in part, on the discovery of a specificrole for MC4-R in body weight regulation. As demonstrated herein, micecompletely lacking MC4-R exhibit significantly increased weight gaincompared to wild-type littermates. In particular, knock-out mice inwhich the gene encoding MC4-R is defective exhibit significant weightgain compared to either MC4-R heterozygous or wild-type femalelittermates. The invention is also based, in part, on the discovery thatthe agouti protein, known to be involved in an obese phenotype whenectopically expressed in mice, binds to the MC4-R.

[0015] The invention relates to assays designed to screen for compoundsor compositions that modulate MC4-R activity, i.e., compounds orcompositions that act as agonists or antagonists of MC4-R, and therebymodulate weight control. To this end, cell-based assays or non-cellbased assays can be used to detect compounds that interact with, e.g.,bind to, a MC4-R extracellular domain (“ECD”). The cell-based assayshave the advantage in that they can be used to identify compounds thataffect MC4-R biological activity (i.e., signal transduction), includingthe identification of compounds that do not interact with a MC4-R ECD,but act on an intracellular component of the signal transduction pathwaymediated by MC4-R.

[0016] The invention also relates to assays designed to screen forcompounds or compositions that modulate MC4-r gene expression. Forexample, cell-based assays, or cell-lysate assays (e.g., in vitrotranscription or translation assays) can be used to screen for compoundsor compositions that modulate MC4-r transcription (e.g., compounds thatmodulate expression, production or activity of transcription factorsinvolved in MC4-r gene expression; polynucleotides that form triplehelical structures with an MC4-r regulatory region and inhibittranscription of the MC4-r gene, etc.). Alternatively, cell-based assaysor cell-lysate assays can be used to screen for compounds orcompositions that modulate translation of MC4-R transcripts (e.g.,antisense and ribozyme molecules).

[0017] In yet another embodiment, the cell-based assays or cell-lysateassays can be used to test polynucleotide constructs designed to modifythe expression of the MC4-r gene in vivo. Such constructs includepolynucleotide constructs designed for gene therapy; e.g., expressionconstructs or gene replacement constructs that place the MC4-r geneunder the control of a strong promoter system, an inducible promotersystem or a constitutive promoter system.

[0018] The invention also encompasses agonists and antagonists of MC4-R,including small molecules, large molecules, and antibodies, as well asnucleotide sequences that can be used to inhibit MC4-r gene expression(e.g., antisense and ribozyme molecules), and gene or regulatorysequence replacement constructs designed to enhance MC4-r geneexpression (e.g., expression constructs that place the MC4-r gene underthe control of a strong promoter system). Such compounds may be used totreat body weight disorders.

[0019] The invention also encompasses the use of such compounds andcompositions, including gene therapy approaches, that modulate MC4-Ractivity or MC4-r gene expression to treat body weight disorders.

[0020] 3.1. Definitions

[0021] The following terms as used herein shall have the meaningindicated.

[0022] MC4-r nucleotides or coding sequences: means DNA sequencesencoding MC4-R mRNA transcripts, MC4-R protein, polypeptide or peptidefragments of MC4-R protein, or MC4-R fusion proteins. MC4-r nucleotidesequences encompass DNA, including genomic DNA (e.g. the MC4-r gene) orcDNA.

[0023] MC4-R means MC4-r gene products, e.g., transcripts and the MC4receptor protein. Polypeptides or peptide fragments of the MC4-R proteinare referred to as MC4-R polypeptides or MC4-R peptides. Fusions ofMC4-R, or MC4-R polypeptides, or peptide fragments to an unrelatedprotein are referred to herein as MC4-R fusion proteins. A functionalMC4-R refers to a protein which binds melanocortin peptides in vivo orin vitro.

[0024] ECD: means “extracellular domain”.

[0025] TM: means “transmembrane domain”.

[0026] CD: means “cytoplasmic domain”.

4. DESCRIPTION OF THE FIGURES

[0027]FIG. 1. Deduced amino acid sequences of the melanocortinreceptors. The serpentine structure of the melanocortin receptorspredicts that the hydrophilic domains located between the TM domains arearranged alternately outside and within the cell to form the ECD (aminoacid residues 1-74, 137-155, 219-231 and 305-316 in FIG. 1) and the CD(amino acid residues 102-112, 178-197, 251-280 and 339-end in FIG. 1) ofthe receptor. The predicted transmembrane domains are denoted byoverbars and Roman numerals, and the four extracellular domains (ECD1,ECD2, ECD3 and ECD4) and four cytoplasmic domains (CD1, CD2, CD3 andCD4) are indicated.

[0028]FIG. 2. Schematic diagram of the construction of the KC4-Rtargeting vector. A) Partial restriction map of the MC4-R locus. B) TheMC4 KO 5′ construct, containing genomic sequences from 3′ of the MC4-Rgene in the vector pJN2. C) The MC4-R RO 5′3′ construct in which genomicsequences from 5′ of the MC4-R gene have been inserted into the MC4 KO5′ construct. D) The MC4-R KO 5′3′ neo construct in which a neoexpression cassette has been inserted between the 5′ and 3′ flankingsequences of the MC4-R gene. The dotted line represents the pJN2 vector.The open box represents the PGK-neo expression cassette, the hatched boxrepresents the MC4-R gene and the arrows indicate the direction oftranscription.

[0029]FIG. 3 Schematic diagram of the gene targeting strategy forinactivation of the MC4-R. (A) Diagram of the MC4-R locus. The hatchedbox represents MC4-R coding sequences, the solid box indicates thelocation of the SacI-SphI probe used for identifying homologousrecombinants. The arrow indicates the direction of transcription of theMC4-R gene. (B) Diagram of the MC4-R targeting construct. The dashedline represents pJN2 plasmid sequences and the arrow indicates thedirection of neo transcription. (C) Diagram of the MC4-R locus followinghomologous recombination with the targeting vector. (D) Predictedrestriction fragment lengths for the wild type and mutated MC4-R locidigested with the indicated enzymes and probed with the SacI-SphI probe.

[0030]FIG. 4 Weight gain of offspring derived from matings of miceheterozygous for MC4-R deletion. Each line represents an individualmouse. The difference in lengths of the lines reflects the difference inages of the various mice being tested. (FIG. 4A) The open arrowsindicate female mice homozygous for MC4-R deletion, unmarked mice arewild type female littermates; (FIG. 4B) The open arrows indicate malemice homozygous for MC4-R deletion, unmarked mice are wild type malelittermates; (FIG. 4C) The closed arrows indicate female miceheterozygous for MC4-R deletion, unmarked mice are wild type femalelittermates; (FIG. 4D) The closed arrows indicate male mice heterozygousfor MC4-R deletion, unmarked mice are wild type male littermates.

[0031]FIG. 5 Sequence of the human MC4-R. Transmembrane domains areunderlined. Amino acid differences in the rat MC4-R are indicatedunderneath the human sequence.

5. DETAILED DESCRIPTION OF THE INVENTION

[0032] The invention described in the subsections below encompassesscreening methods (e.g., assays) for the identification of compoundswhich affect weight modulation. The invention also encompasses agonistsand antagonists of MC4-R, including small molecules, large molecules,and antibodies, as well as nucleotide sequences that can be used toinhibit MC4-r gene expression (e.g., antisense and ribozyme molecules),and gene or regulatory sequence replacement constructs designed toenhance MC4-r gene expression (e.g., expression constructs that placethe MC4-r gene under the control of a strong promoter system). Suchcompounds may be used to treat body weight disorders. In particular,cellular and non-cellular assays are described that can be used toidentify compounds that interact with the MC4-R, e.g., modulate theactivity of the MC4-R and/or bind to the MC4-R. The cell based assayscan be used to identify compounds or compositions that affect thesignal-transduction activity of the MC4-R, whether they bind to theMC4-R or act on intracellular factors involved in the MC4-R signaltransduction pathway. Such cell-based assays of the invention utilizecells, cell lines, or engineered cells or cell lines that express theMC4-R. The cells can be further engineered to incorporate a reportermolecule linked to the signal transduced by the activated MC4-R to aidin the identification of compounds that modulate MC4-R signallingactivity.

[0033] The invention also encompasses the use of cell-based assays orcell-lysate assays (e.g., in vitro transcription or translation assays)to screen for compounds or compositions that modulate MC4-r geneexpression. To this end, constructs containing a reporter sequencelinked to a regulatory element of the MC4-r gene can be used inengineered cells, or in cell lysate extracts, to screen for compoundsthat modulate the expression of the reporter gene product at the levelof transcription. For example, such assays could be used to identifycompounds that modulate the expression or activity of transcriptionfactors involved in MC4-r gene expression, or to test the activity oftriple helix polynucleotides. Alternatively, engineered cells ortranslation extracts can be used to screen for compounds (includingantisense and ribozyme constructs) that modulate the translation ofMC4-R mRNA transcripts, and therefore, affect expression of the MC4-R.

[0034] The invention also encompasses MC4-R proteins, polypeptides(including soluble MC4-R polypeptides or peptides) and MC4-R fusionproteins for use in non-cell based screening assays, for use ingenerating antibodies, for diagnostics and therapeutics. The MC4-R ispredicted to be a serpentine receptor that traverses the membrane seventimes, resulting in four extra-cellular domains (ECDS) and four cellulardomains (CDs) (see FIG. 1). Peptides corresponding to each ECD, or apolypeptide composed of two or more of the four ECDs linked together canbe engineered as described in Section 5.3.1, infra. Alternatively, suchpeptides or polypeptides can be fused to a heterologous protein, e.g., areporter, an Ig Fc region, etc., to yield a fusion protein. Suchpeptides, polypeptides and fusion proteins can be used in the non-cellbased assays for screening compounds that interact with, e.g., modulatethe activity of the MC4-R and or bind to the MC4-R.

[0035] MC4-R protein products can be used to treat weight disorders suchas obesity, anorexia or cachexia. Such MC4-R protein products includebut are not limited to soluble derivatives such as peptides orpolypeptides corresponding to one or more MC4-R ECDs; truncated MC4-Rpolypeptides lacking one or more ECD or TM; and MC4-R fusion proteinproducts (especially MC4-R-Ig fusion proteins, i.e., fusions of theMC4-R or a domain of the MC4-R, to an IgFc domain). Alternatively,antibodies to the MC4-R or anti-idiotypic antibodies that mimic theMC4-R (including Fab fragments), antagonists or agonists (includingcompounds that modulate signal transduction which may act on downstreamtargets in the MC4-R signal transduction pathway) can be used to treatbody weight disorders such as obesity, anorexia or cachexia.

[0036] For example, the administration of an effective amount of solubleMC4-R polypeptide, or an MC4-R fusion protein (e.g., MC4-R ECD-IgFc) oran anti-idiotypic antibody (or its Fab) that mimics the MC4-R ECD wouldinteract with and thereby “mop up” or “neutralize” endogenous MC4-Rligand, and prevent or reduce binding and receptor activation, leadingto weight gain. In yet another approach, nucleotide constructs encodingsuch MC4-R products can be used to genetically engineer host cells toexpress such MC4-R products in vivo; these genetically engineered cellscan function as “bioreactors” in the body delivering a continuous supplyof the MC4-R, MC4-R peptide, soluble MC4-R polypeptide, or MC4-R fusionprotein that will “mop up” or neutralize MC4-R ligand. “Gene therapy”approaches for the modulation of MC4-R expression and/or activity in thetreatment of body weight disorders are within the scope of theinvention. For example, nucleotide constructs encoding functionalMC4-Rs, mutant MC4-Rs, as well as antisense and ribozyme molecules canbe used to modulate MC4-r expression.

[0037] The invention also encompasses pharmaceutical formulations andmethods for treating body weight disorders.

[0038] 5.1. The Role of MC4-R in the Regulation of Body Weight

[0039] The specific role of the MC4-R protein in vivo was investigatedby engineering MC4-R “knock out” mice in which most of the endogenousMC4-R gene coding sequence was deleted, thereby creating mice which areunable to produce functional MC4-R protein. Unlike MC-Ragonist/antagonist studies which are complicated because each of the MCreceptors, rather than just MC4-R, can be affected, this specificelimination of only MC4-R function allowed an evaluation of thebiological function of MC4-R.

[0040] In order to produce the MC4-R knock out mice, human MC4-r genesequences were utilized to isolate and clone the murine MC4-r gene. Amurine MC4-r targeting construct was then generated which was designedto delete the majority of the murine MC4-r coding sequence uponhomologous recombination with the endogenous murine MC4-r gene.

[0041] Embryonic stem (ES) cells containing the disrupted MC4-r genewere produced, isolated and microinjected into murine blastocysts toyield mice chimeric for cells containing a disrupted MC4-r gene.Offspring of the chimeric mice resulting from germline transmission ofthe ES genome were obtained and animals heterozygous for the disruptedMC4-R were identified.

[0042] In order to assess the role of MC4-R in vivo, the animalsheterozygous for the MC4-r disrupted gene were bred together, producinglitters containing wild-type mice, mice heterozygous for the MC4-rmutation and mice homozygous for the MC4-R mutation. The weight gain ofthe animals was monitored regularly. Homozygous null MC4-R mutantsshowed an increase in weight compared to mice heterozygous for MC4-Rdeletion and wild type mice as early as 25 days of age. By 54-58 days ofage, MC4-R deficient mice exhibited, on average, a 55-70% greater weightrelative to wild type mice, and an approximately 50% greater weightcompared to mice heterozygous for the MC4-R deletion.

[0043] The knock out experiments described herein represent definitiveevidence of the role of MC4-R in weight regulation. The experimentaldesign does not rely on the relationship, if any, of the agouti ligandfor the characterization of the functional role of the MC4-R.

[0044] 5.2. Screening Assays for Drugs Useful in Regulation of BodyWeight

[0045] At least three different assay systems, described in thesubsections below, can be designed and used to identify compounds orcompositions that modulate MC4-R activity or MC4-r gene expression, andtherefore, modulate weight control.

[0046] The systems described below may be formulated into kits. To thisend, the MC4-R or cells expressing the MC4-R can be packaged in avariety of containers, e.g., vials, tubes, microtitre well plates,bottles, and the like. Other reagents can be included in separatecontainers and provided with the kit; e.g., positive controls samples,negative control samples, melanocortin peptides (including but notlimited to αMSH and ACTH derivatives), buffers, cell culture media, etc.

[0047] 5.2.1. Cell-Based Assays

[0048] In accordance with the invention, a cell-based assay system canbe used to screen for compounds that modulate the activity of the MC4-Rand thereby, modulate body weight. To this end, cells that endogenouslyexpress MC4-R can be used to screen for compounds. Alternatively, celllines, such as 293 cells, COS cells, CHO cells, fibroblasts, and thelike, genetically engineered to express the MC4-R can be used forscreening purposes. Preferably, host cells genetically engineered toexpress a functional receptor that responds to activation bymelanocortin peptides can be used as an endpoint in the assay; e.g., asmeasured by a chemical, physiological, biological, or phenotypic change,induction of a host cell gene or a reporter gene, change in cAMP levels,adenylyl cyclase activity, host cell G protein activity, extracellularacidification rate, host cell kinase activity, proliferation,differentiation, etc.

[0049] To be useful in screening assays, the host cells expressingfunctional MC4-R should give a significant response to MC4-R ligand,preferably greater than 5-fold induction over background. Host cellsshould preferably possess a number of characteristics, depending on thereadout, to maximize the inductive response by melanocortin peptides,for example, for detecting a strong induction of a CRE reporter gene:(a) a low natural level of cAMP, (b) G proteins capable of interactingwith the MC4-R, (c) a high level of adenylyl cyclase, (d) a high levelof protein kinase A, (e) a low level of phosphodiesterases, and (f) ahigh level of cAMP response element binding protein would beadvantageous. To increase response to melanocortin peptide, host cellscould be engineered to express a greater amount of favorable factors ora lesser amount of unfavorable factors. In addition, alternativepathways for induction of the CRE reporter could be eliminated to reducebasal levels.

[0050] In utilizing such cell systems, the cells expressing themelanocortin receptor are exposed to a test compound or to vehiclecontrols (e.g., placebos). After exposure, the cells can be assayed tomeasure the expression and/or activity of components of the signaltransduction pathway of the melanocortin receptor, or the activity ofthe signal transduction pathway itself can be assayed. For example,after exposure, cell lysates can be assayed for induction of cAMP. Theability of a test compound to increase levels of cAMP, above thoselevels seen with cells treated with a vehicle control, indicates thatthe test compound induces signal transduction mediated by themelanocortin receptor expressed by the host cell. In screening forcompounds that may act as antagonists of MC4-R, it is necessary toinclude ligands that activate the MC4-R, e.g., α-MSH, β-MSH or ACTH, totest for inhibition of signal transduction by the test compound ascompared to vehicle controls.

[0051] In a specific embodiment of the invention, constructs containingthe cAMP responsive element linked to any of a variety of differentreporter genes may be introduced into cells expressing the melanocortinreceptor. Such reporter, genes may include but is not limited tochloramphenicol acetyltransferase (CAT), luciferase, GUS, growthhormone, or placental alkaline phosphatase (SEAP). Following exposure ofthe cells to the test compound, the level of reporter gene expressionmay be quantitated to determine the test compound's ability to regulatereceptor activity. Alkaline phosphatase assays are particularly usefulin the practice of the invention as the enzyme is secreted from thecell. Therefore, tissue culture supernatant may be assayed for secretedalkaline phosphatase. In addition, alkaline phosphatase activity may bemeasured by calorimetric, bioluminescent or chemilumenscent assays suchas those described in Bronstein, I. et al. (1994, Biotechniques 17:172-177). Such assays provide a simple, sensitive easily automatabledetection system for pharmaceutical screening.

[0052] When it is desired to discriminate between the melanocortinreceptors and to identify compounds that selectively agonize orantagonize the MC4-R, the assays described above should be conductedusing a panel of host cells, each genetically engineered to express oneof the melanocortin receptors (MC1-R through MC5-R). Expression of thehuman melanocortin receptors is preferred for drug discovery purposes.To this end, host cells can be genetically engineered to express any ofthe amino acid sequences shown for melanocortin receptors 1 through 5 inFIG. 1. The cloning and characterization of each receptor has beendescribed: MC1-R and MC2-R (Mountjoy., 1992, Science 257: 1248-1251;Chhajlani & Wikberg, 1992 FEBS Lett. 309: 417-420); MC3-R(Roselli-Rehfuss et al., 1993, Proc. Natl. Acad. Sci., USA 90:8856-8860; Gantz et al., 1993, J. Biol. Chem. 268: 8246-8250); MC4-R(Gantz et al., 1993, J. Biol. Chem. 268: 15174-15179; Mountjoy et al.,1994, Mol. Endo. 8: 1298-1308); and MC5-R (Chhajlani et al., 1993,Biochem. Biophys. Res. Commun. 195: 866-873; Gantz et al., 1994,Biochem. Biophys. Res. Commun. 200; 1214-1220), each of which isincorporated by reference herein in its entirety. Thus, each of theforegoing sequences can be utilized to engineer a cell or cell line thatexpresses one of the melanocortin receptors for use in screening assaysdescribed herein. To identify compounds that specifically or selectivelyregulate MC4-R activity, the activation, or inhibition of MC4-Ractivation is compared to the effect of the test compound on the othermelanocortin receptors.

[0053] Alternatively, if the host cells express more than onemelanocortin peptide receptor, the background signal produced by thesereceptors in response to melanocortin peptides must be “subtracted” fromthe signal (see Gantz et al., supra). The background response producedby these non-MC4-R melanocortin receptors can be determined by a numberof methods, including elimination of MC4-R activity by antisense,antibody or antagonist. In this regard, it should be noted that wildtype CHO cells demonstrate a small endogenous response to melanocortinpeptides which must be subtracted from background. Alternatively,activity contributed from other melanocortin receptors could beeliminated by activating host cells with a MC4-R-specific ligand, orincluding specific inhibitors of the other melanocortin receptors.

[0054] 5.2.2. Non-Cell Based Assays

[0055] In addition to cell based assays, non-cell based assay systemsmay be used to identify compounds that interact with, e.g., bind toMC4-R. Such compounds may act as antagonists or agonists of MC4-Ractivity and may be used in the treatment of body weight disorders.

[0056] Isolated membranes may be used to identify compounds thatinteract with MC4-R. For example, in a typical experiment using isolatedmembranes, 293 cells may be genetically engineered to express the MC4-R.Membranes can be harvested by standard techniques and used in an invitro binding assay. ¹²⁵I-labelled ligand (e.g., ¹²⁵I-labelled α-MSH,β-MSH, or ACTH) is bound to the membranes and assayed for specificactivity; specific binding is determined by comparison with bindingassays performed in the presence of excess unlabelled ligand.

[0057] To identify MC4-R ligands, membranes are incubated with labelledligand in the presence or absence of test compound. Compounds that bindto the receptor and compete with labelled ligand for binding to themembranes reduced the signal compared to the vehicle control samples.

[0058] Alternatively, soluble MC4-R may be recombinantly expressed andutilized in non-cell based assays to identify compounds that bind toMC4-R. The recombinantly expressed MC4-R polypeptides or fusion proteinscontaining one or more of the ECDs of MC4-R prepared as described inSection 5.3.1, infra, can be used in the non-cell based screeningassays.

[0059] Alternatively, peptides corresponding to one or more of the CDsof MC4-R, or fusion proteins containing one or more of the CDs of MC4-Rcan be used in non-cell based assay systems to identify compounds thatbind to the cytoplasmic portion of the MC4-R; such compounds may beuseful to modulate the signal transduction pathway of the MC4-R. Innon-cell based assays the recombinantly expressed MC4-R is attached to asolid substrate such as a test tube, microtitre well or a column, bymeans well known to those in the art (see Ausubel et al., supra). Thetest compounds are then assayed for their ability to bind to the MC4-R.

[0060] In one aspect of the invention the screens may be designed toidentify compounds that antagonize the interaction between MC4-R andMC4-R ligands such as α-MSH, β-MSH and ACTH. In such screens, the MC4-Rligands are labelled and test compounds can be assayed for their abilityto antagonize the binding of labelled ligand to MC4-R.

[0061] 5.2.3. Assays for Compounds or Compositions That ModulateExpression of the MC4-R

[0062] In vitro cell based assays may be designed to screen forcompounds that regulate MC4-R expression at either the transcriptionalor translational level.

[0063] In one embodiment, DNA encoding a reporter molecule can be linkedto a regulatory element of the MC4-r gene and used in appropriate intactcells, cell extracts or lysates to identify compounds that modulateMC4-r gene expression. Appropriate cells or cell extracts are preparedfrom any cell type that normally expresses the MC4-r gene, therebyensuring that the cell extracts contain the transcription factorsrequired for in vitro or in vivo transcription. The screen can be usedto identify compounds that modulate the expression of the reporterconstruct. In such screens, the level of reporter gene expression isdetermined in the presence of the test compound and compared to thelevel of expression in the absence of the test compound.

[0064] To identify compounds that regulate MC4-R translation, cells orin vitro cell lysates containing MC4-R transcripts may be tested formodulation of MC4-R mRNA translation. To assay for inhibitors of MC4-Rtranslation, test compounds are assayed for their ability to modulatethe translation of MC4R mRNA in in vitro translation extracts.

[0065] Compounds that decrease the level of MC4-R expression, either atthe transcriptional or translational level, may be useful for treatmentof body weight disorders such as anorexia and cachexia. In contrast,those compounds that increase the expression of MC4-R may be useful fortreatment of disorders such as obesity.

[0066] 5.2.4. Compounds that can be Screened in Accordance with theInvention

[0067] The assays described above can identify compounds which affectMC4-R activity. For example, compounds that affect MC4-R activityinclude but are not limited to compounds that bind to the MC4-R, inhibitbinding of the natural ligand, and either activate signal transduction(agonists) or block activation (antagonists), and compounds that bind tothe natural ligand of the MC4-R and neutralize ligand activity.Compounds that affect MC4-r gene activity (by affecting MC4-r geneexpression, including molecules, e.g., proteins or small organicmolecules, that affect transcription or interfere with splicing eventsso that expression of the full length or the truncated form of the MC4-Rcan be modulated) can also be identified on the screens of theinvention. However, it should be noted that the assays described canalso identify compounds that modulate MC4-R signal transduction (e.g.,compounds which affect downstream signalling events, such as inhibitorsor enhancers of G protein activities which participate in transducingthe signal activated by ligand binding to the MC4-R). The identificationand use of such compounds which affect signalling events downstream ofMC4-R and thus modulate effects of MC4-R on the development of bodyweight disorders are within the scope of the invention. In someinstances, G protein-coupled receptors response has been observed tosubside, or become desensitized with prolonged exposure to ligand. In anembodiment of the invention assays may be utilized to identify compoundsthat block the desensitization of the MC4-receptor, such compounds maybe used to sustain the activity of the MC4-receptor, such compounds maybe used to sustain the activity of the MC4-R receptor. Such compoundscan be used as part of a therapeutic method for the treatment of bodyweight disorders.

[0068] The compounds which may be screened in accordance with theinvention include, but are not limited to peptides, antibodies andfragments thereof, and other organic compounds (e.g., peptidomimetics)that bind to the ECD of the MC4-R and either mimic the activitytriggered by the natural ligand (i.e., agonists) or inhibit the activitytriggered by the natural ligand (i.e., antagonists); as well aspeptides, antibodies or fragments thereof, and other organic compoundsthat include the ECD of the MC4-R (or a portion thereof) and bind to and“neutralize” natural ligand.

[0069] Compounds may include, but are not limited to, peptides such as,for example, soluble peptides, including but not limited to members ofrandom peptide libraries; (see, e.g., Lam, K. S. et al., 1991, Nature354:82-84; Houghten, R. et al., 1991, Nature 354:84-86), andcombinatorial chemistry-derived molecular library made of D- and/orL-configuration amino acids, phosphopeptides (including, but not limitedto, members of random or partially degenerate, directed phosphopeptidelibraries; see, e.g., Songyang, Z. et al., 1993, Cell 72:767-778),antibodies (including, but not limited to, polyclonal, monoclonal,humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb,F(ab′)₂ and FAb expression library fragments, and epitope-bindingfragments thereof), and small organic or inorganic molecules.

[0070] Other compounds which can be screened in accordance with theinvention include but are not limited to small organic molecules thatare able to cross the blood-brain barrier, gain entry into anappropriate cell and affect the expression of the MC4-R gene or someother gene involved in the MC4-R signal transduction pathway (e.g., byinteracting with the regulatory region or transcription factors involvedin gene expression); or such compounds that affect the activity of theMC4-R or the activity of some other intracellular factor involved in theMC4-R signal transduction pathway, such as, for example, the MC4-Rassociated G protein.

[0071] Computer modelling and searching technologies permitidentification of compounds, or the improvement of already identifiedcompounds, that can modulate MC4-R expression or activity. Havingidentified such a compound or composition, the active sites or regionsare identified. Such active sites might typically be ligand bindingsites. The active site can be identified using methods known in the artincluding, for example, from the amino acid sequences of peptides, fromthe nucleotide sequences of nucleic acids, or from study of complexes ofthe relevant compound or composition with its natural ligand. In thelatter case, chemical or X-ray crystallographic methods can be used tofind the active site by finding where on the factor the complexed ligandis found.

[0072] Next, the three dimensional geometric structure of the activesite is determined. This can be done by known methods, including X-raycrystallography, which can determine a complete molecular structure. Onthe other hand, solid or liquid phase NMR can be used to determinecertain intra-molecular distances. Any other experimental method ofstructure determination can be used to obtain partial or completegeometric structures. The geometric structures may be measured with acomplexed ligand, natural or artificial, which may increase the accuracyof the active site structure determined.

[0073] If an incomplete or insufficiently accurate structure isdetermined, the methods of computer based numerical modelling can beused to complete the structure or improve its accuracy. Any recognizedmodelling method may be used, including parameterized models specific toparticular biopolymers such as proteins or nucleic acids, moleculardynamics models based on computing molecular motions, statisticalmechanics models based on thermal ensembles, or combined models. Formost types of models, standard molecular force fields, representing theforces between constituent atoms and groups, are necessary, and can beselected from force fields known in physical chemistry. The incompleteor less accurate experimental structures can serve as constraints on thecomplete and more accurate structures computed by these modelingmethods.

[0074] Finally, having determined the structure of the active site,either experimentally, by modeling, or by a combination, candidatemodulating compounds can be identified by searching databases containingcompounds along with information on their molecular structure. Such asearch seeks compounds having structures that match the determinedactive site structure and that interact with the groups defining theactive site. Such a search can be manual, but is preferably computerassisted. These compounds found from this search are potential MC4-Rmodulating compounds.

[0075] Alternatively, these methods can be used to identify improvedmodulating compounds from an already known modulating compound orligand. The composition of the known compound can be modified and thestructural effects of modification can be determined using theexperimental and computer modelling methods described above applied tothe new composition. The altered structure is then compared to theactive site structure of the compound to determine if an improved fit orinteraction results. In this manner systematic variations incomposition, such as by varying side groups, can be quickly evaluated toobtain modified modulating compounds or ligands of improved specificityor activity.

[0076] Further experimental and computer modeling methods useful toidentify modulating compounds based upon identification of the activesites of MC4-R, and related transduction and transcription factors willbe apparent to those of skill in the art.

[0077] Examples of molecular modelling systems are the CHARm and QUANTAprograms (Polygen Corporation, Waltham, Mass.). CHARnm performs theenergy minimization and molecular dynamics functions. QUANTA performsthe construction, graphic modelling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other.

[0078] A number of articles review computer modelling of drugsinteractive with specific proteins, such as Rotivinen, et al.) 1988,Acta Pharmaceutical Fennica 97:159-166); Ripka (1988 New Scientist54-57); McKinaly and Rossmann (1989, Annu. Rev. Pharmacol. Toxiciol.29:111-122); Perry and Davies, OSAR: Quantitative Structure-ActivityRelationships in Drug Design pp. 189-193 Alan R. Liss, Inc. 1989; Lewisand Dean (1989,. Proc. R. Soc. Lond. 236:125-140 and 141-162); and, withrespect to a model receptor for nucleic acid components, Askew, et al.(1989, J. Am. Chem. Soc. 111:1082-1090). Other computer programs thatscreen and graphically depict chemicals are available from companiessuch as BioDesign, Inc. (Pasadena, Calif.), Allelix, Inc. (Mississauga,Ontario, Canada), and Hypercube, Inc. (Cambridge, Ontario). Althoughthese are primarily designed for application to drugs specific toparticular proteins, they can be adapted to design of drugs specific toregions of DNA or RNA, once that region is identified.

[0079] Although described above with reference to design and generationof compounds which could alter binding, one could also screen librariesof known compounds, including natural products or synthetic chemicals,and biologically active materials, including proteins, for compoundswhich are inhibitors or activators.

[0080] Compounds identified via assays such as those described hereinmay be useful, for example, in elaborating the biological function ofthe MC4-R gene product, and for ameliorating body weight disorders.Assays for testing the efficacy of compounds identified in the cellularscreen can be tested in animal model systems for body weight disorders.Such animal models may be used as test substrates for the identificationof drugs, pharmaceuticals, therapies and interventions which may beeffective in treating such disorders. For example, animal models may beexposed to a compound, suspected of exhibiting an ability to amelioratebody weight disorder symptoms, at a sufficient concentration and for atime sufficient to elicit such an amelioration of body weight disordersymptoms in the exposed animals. The response of the animals to theexposure may be monitored by assessing the reversal of disordersassociated with body weight disorders such as obesity. With regard tointervention, any treatments which reverse any aspect of body weightdisorder-like symptoms should be considered as candidates for human bodyweight disorder therapeutic intervention. Dosages of test agents may bedetermined by deriving dose-response curves, as discussed in Section5.5, below.

[0081] To this end, transgenic animals that express the human MC4-r geneproducts can be used. Animals of any species, including, but not limitedto, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, andnon-human primates, e.g., baboons, monkeys, and chimpanzees may be usedto generate MC4-R transgenic animals.

[0082] Any technique known in the art may be used to introduce the humanMC4-r transgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to pronuclearmicroinjection (Hoppe, P. C. and Wagner, 1989, U.S. Pat. No. 4,873,191);retrovirus mediated gene transfer into germ lines (Van der Putten etal., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); gene targeting inembryonic stem cells (Thompson et al., 1989, Cell 56:313-321);electroporation of embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814); andsperm-mediated gene transfer (Lavitrano et al., 1989, Cell 57:717-723);etc. For a review of such techniques, see Gordon, 1989, TransgenicAnimals, Intl. Rev. Cytol. 115:171-229, which is incorporated byreference herein in its entirety.

[0083] The present invention provides for transgenic animals that carrythe MC4-r transgene in all their cells, as well as animals which carrythe transgene in some, but not all their cells, i.e., mosaic animals.The transgene may be integrated as a single transgene or in concatamers,e.g., head-to-head tandems or head-to-tail tandems. The transgene mayalso be selectively introduced into and activated in a particular celltype by following, for example, the teaching of Lasko et al. (Lasko, M.et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6232-6236). The regulatorysequences required for such a cell-type specific activation will dependupon the particular cell type of interest, and will be apparent to thoseof skill in the art. When it is desired that the MC4-r transgene beintegrated into the chromosomal site of the endogenous MC4-r gene, genetargeting is preferred. Briefly, when such a technique is to beutilized, vectors containing nucleotide sequences homologous to theendogenous MC4-r gene and/or sequences flanking the gene are designedfor the purpose of integrating, via homologous recombination withchromosomal sequences, into and disrupting the function of theendogenous MC4-r gene. The transgene may also be selectively expressedin a particular cell type with concomitant inactivation of theendogenous MC4-r gene in only that cell type, by following, for example,the teaching of Gu et al. (Gu, et al., 1994, Science 265: 103-106). Theregulatory sequences required for such a cell-type specificrecombination will depend upon the particular cell type of interest, andwill be apparent to those of skill in the art.

[0084] Once founder animals have been generated, standard techniquessuch as Southern blot analysis or PCR techniques are used to analyzeanimal tissues to determine whether integration of the transgene hastaken place. The level of mRNA expression of the transgene in thetissues of the founder animals may also be assessed using techniqueswhich include but are not limited to Northern blot analysis of tissuesamples obtained from the animal, in situ hybridization analysis, andRT-PCR. Samples of MC4-R gene-expressing tissue, may also be evaluatedimmunocytochemically using antibodies specific for the MC4-R transgeneproduct.

[0085] 5.3. MC4-R Proteins, Polypeptides, and Antibodies

[0086] MC4-R protein, polypeptides and peptide fragments, mutated,truncated or deleted forms of the MC4-R and/or MC4-R fusion proteins canbe prepared for a variety of uses, including but not limited to thegeneration of antibodies, as reagents in diagnostic assays, theidentification of other cellular gene products involved in theregulation of body weight, as reagents in assays for screening forcompounds that can be used in the treatment of body weight disorders,and as pharmaceutical reagents useful in the treatment of body weightdisorders related to the MC4-R.

[0087] 5.3.1. Production of MC4-R Polypeptides

[0088] The deduced amino acid sequences of the melanocortin receptors,including MC4-R, are shown in FIG. 1, where predicted transmembranedomains are denoted by overbars and Roman numerals, and the fourextracellular domains (ECD1, ECD2, ECD3, and ECD4) and the fourcytoplasmic domains (CD1, CD2, CD3 and CD4) are indicated. Theserpentine structure of the melanocortin receptors predicts that thehydrophilic domains located between the TM domains are arrangedalternately outside and within the cell to form the ECD (amino acidresidues 1-74-, 137-155, 219-231 and 305-316 in FIG. 1) and the CD(amino acid residues 102-112, 178-197, 251-280 and 339-end in FIG. 1) ofthe receptor. Peptides corresponding to one or more domains of the MC4-R(e.g., ECDS, TMs or CDs), truncated or deleted MC4-R (e.g., MC4-R inwhich one or more of the ECDs, TMs and/or CDs is deleted) as well asfusion proteins in which the full length MC4-R, an MC4-R peptide ortruncated MC4-R is fused to an unrelated protein are also within thescope of the invention. Such soluble peptides, proteins, fusionproteins, or antibodies (including anti-idiotypic antibodies) that bindto and “neutralize” circulating natural ligand for the MC4-R, can beused as described in Section 5.5, infra, to effectuate weight gain. Tothis end, peptides corresponding to individual ECDs of MC4-R, solubledeletion mutants of MC4-R (e.g., ATM mutants), or the entire MC4-R ECD(engineered by linking the four ECDs together as described below) can befused to another polypeptide (e.g., an IgFc polypeptide). Fusion of theMC4-R or the MC4-R ECD to an IgFc polypeptide should not only increasethe stability of the preparation, but will increase the half-life andactivity of the MC4-R-Ig fusion protein in vivo. The Fc region of the Igportion of the fusion protein may be further modified to reduceimmunoglobulin effector function.

[0089] Such peptides, polypeptides., and fusion proteins can be preparedby recombinant DNA techniques. For example, nucleotide sequencesencoding one or more of the four domains of the ECD of the serpentineMC4-R can be synthesized or cloned and ligated together to encode asoluble ECD of the MC4-R. The DNA sequence encoding one or more of thefour ECDs (ECD1-4 in FIG. 1) can be ligated together directly or via alinker oligonucleotide that encodes a peptide spacer. Such linkers mayencode flexible, glycine-rich amino acid sequences thereby allowing thedomains that are strung together to assume a conformation that can bindMC4-R ligands. Alternatively, nucleotide sequences encoding individualdomains within the ECD can be used to express MC4R peptides.

[0090] A variety of host-expression vector systems may be utilized toexpress nucleotide sequences encoding the appropriate regions of theMC4-R to produce such polypeptides. Where the resulting peptide orpolypeptide is a soluble derivative (e.g., peptides corresponding to theECDs; truncated or deleted in which the TMs and/or CDs are deleted) thepeptide or polypeptide can be recovered from the culture media. Wherethe polypeptide or protein is not secreted, the MC4-R product can berecovered from the host cell itself.

[0091] The host-expression vector systems also encompass engineered hostcells that express the MC4-R or functional equivalents in situ, i.e.,anchored in the cell membrane. Purification or enrichment of the MC4-Rfrom such expression systems can be accomplished using appropriatedetergents and lipid micelles and methods well known to those skilled inthe art. However, such engineered host cells themselves may be used insituations where it is important not only to retain the structural andfunctional characteristics of the MC4-R, but to assess biologicalactivity, e.g., in drug screening assays.

[0092] The host-expression vector systems that may be used for purposesof the invention include but are not limited to microorganisms such asbacteria (e.g., E. coli, B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining MC4-R nucleotide sequences; yeast (e.g., Saccharomyces,Pichia) transformed with recombinant yeast expression vectors containingthe MC4-R nucleotide sequences; insect cell systems infected withrecombinant virus expression vectors (e.g., baculovirus) containing theMC4-R sequences; plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower, mosaic virus, CaMV; tobaccomosaic virus, TMV) or transformed with recombinant plasmid expressionvectors (e.g., Ti plasmid) containing MC4-R nucleotide sequences; ormammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

[0093] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the MC4-Rgene product being expressed. For example, when a large quantity of sucha protein is to be produced, for the generation of pharmaceuticalcompositions of MC4-R protein or for raising antibodies to the MC4-Rprotein, for example, vectors which direct the expression of high levelsof fusion protein products that are readily purified may be desirable.Such vectors include, but are not limited, to the E. coli expressionvector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the MC4-Rcoding sequence may be ligated individually into the vector in framewith the lacZ coding region so that a fusion protein is produced; pINvectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; VanHeeke & Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the like.PGEX vectors may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The PGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned targetgene product can be released from the GST moiety.

[0094] Alternatively, any fusion protein may be readily purified byutilizing an antibody specific for the fusion protein being expressed.For example, a system described by Janknecht et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-8976). In this system, the gene of interest is subcloned into avaccinia recombination plasmid such that the gene's open reading frameis translationally fused to an Amino-terminal tag consisting of sixhistidine residues. Extracts from cells infected with recombinantvaccinia virus are loaded onto Ni² ⁺-nitriloacetic acid-agarose columnsand histidine-tagged proteins are selectively eluted withimidazole-containing buffers.

[0095] In an insect system, Autoarapha californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign genes. The virusgrows in Spodotera frugiperda cells. The MC4-R coding sequence may becloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter). Successful insertion ofMC4-R gene coding sequence will result in inactivation of the polyhedringene and production of non-occluded recombinant virus (i.e., viruslacking the proteinaceous coat coded for by the polyhedrin gene). Therecombinant viruses are then used to infect cells in which the insertedgene is expressed. (E.g., see Smith et al., 1983, J. Virol. 46: 584;Smith, U.S. Pat. No. 4,215,051).

[0096] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the MC4-R nucleotide sequence of interest may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericgene may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing the MC4-R gene product in infectedhosts. (E.g., See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA81:3655-3659). Specific initiation signals may also be required forefficient translation of inserted MC4-R nucleotide sequences. Thesesignals include the ATG initiation codon and adjacent sequences. Incases where an entire MC4-R gene or cDNA, including its own initiationcodon and adjacent sequences, is inserted into the appropriateexpression vector, no additional translational control signals may beneeded. However, in cases where only a portion of the MC4-R codingsequence is inserted, exogenous translational control signals,including, perhaps, the ATG initiation codon, must be provided.Furthermore, the initiation codon must be in frame with the readingframe of the desired coding sequence to ensure translation of the entireinsert. These exogenous translational control signals and initiationcodons can be of a variety of origins, both natural and synthetic. Theefficiency of expression may be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (SeeBittner et al., 1987, Methods in Enzymol. 153:516-544).

[0097] In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed.Accordingly, eukaryotic host cells which possess the cellular machineryfor proper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK,293, 3T3 and WI38 cell lines.

[0098] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress the MC4-R sequences described above may be engineered. Ratherthan using expression vectors which contain viral origins ofreplication, host cells can be transformed with DNA controlled byappropriate expression control elements (e.g., promoter, enhancersequences, transcription terminators, polyadenylation sites, etc.), anda selectable marker. Following the introduction of the foreign DNA,engineered cells may be allowed to grow for 1-2 days in an enrichedmedia, and then are switched to a selective media. The selectable markerin the recombinant plasmid confers resistance to the selection andallows cells to stably integrate the plasmid into their chromosomes andgrow to form foci which in turn can be cloned and expanded into celllines. This method may advantageously be used to engineer cell lineswhich express the MC4-R gene product. Such engineered cell lines may beparticularly useful in screening and evaluation of compounds that affectthe endogenous activity of the MC4-R gene product.

[0099] A number of selection systems may be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler, et al.,1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), andadenine phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817)genes can be employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigler,et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc.Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro, whichconfers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147).

[0100] 5.3.2. Antibodies to MC4-R polypeptides

[0101] Antibodies that specifically recognize one or more epitopes ofMC4-R, or epitopes of conserved variants of MC4-R, or peptide fragmentsof the MC4-R are also encompassed by the invention. Such antibodiesinclude but are not limited to polyclonal antibodies, monoclonalantibodies (mAbs), humanized or chimeric antibodies, single chainantibodies, Fab fragments, F(ab′)₂ fragments, fragments produced by aFab expression library, anti-idiotypic (anti-Id) antibodies, andepitope-binding fragments of any of the above.

[0102] The antibodies of the invention may be used, for example, in thedetection of the MC4-R in a biological sample and may, therefore, beutilized as part of a diagnostic or prognostic technique wherebypatients may be tested for abnormal amounts of MC4-R. Such antibodiesmay also be utilized in conjunction with, for example, compoundscreening schemes, as described, above, for the evaluation of the effectof test compounds on expression and/or activity of the MC4-R geneproduct. Additionally, such antibodies can be used in conjunction withthe gene therapy techniques described, below, e.g., to evaluate thenormal and/or engineered MC4-R-expressing cells prior to theirintroduction into the patient. Such antibodies may additionally be usedas a method for the inhibition of abnormal MC4-R activity. Thus, suchantibodies may, therefore, be utilized as part of weight disordertreatment methods.

[0103] For the production of antibodies, various host animals may beimmunized by injection with the MC4-R, an MC4-R peptide (e.g., onecorresponding the a functional domain of the receptor, such as ECD, TMor CD), truncated MC4-R polypeptides (MC4-R in which one or moredomains, e.g., the TM or CD, has been deleted), functional equivalentsof the MC4-R or mutants of the MC4-R. Such host animals may include butare not limited to rabbits, mice, hamsters and rats, to name but a few.Various adjuvants may be used to increase the immunological response,depending on the host species, including but not limited to Freund's(complete and incomplete), mineral gels such as aluminum hydroxide.,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum. Polyclonalantibodies are heterogeneous populations of antibody molecules derivedfrom the sera of the immunized animals.

[0104] Monoclonal antibodies, which are homogeneous populations ofantibodies to a particular antigen, may be obtained by any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497;and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique(Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc.Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique(Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R.Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulinclass including IgG, IgM, IgE, IgA, IgD and any subclass thereof. Thehybridoma producing the mAb of this invention may be cultivated in vitroor in vivo. Production of high titers of mabs in vivo makes this thepresently preferred method of production.

[0105] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine nAb and a human immunoglobulin constantregion.

[0106] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; and Ward et al., 1989, Nature 334:544-546) can be adaptedto produce single chain antibodies against MC4-R gene products. Singlechain antibodies are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge, resulting in asingle chain polypeptide.

[0107] Antibody fragments which recognize specific epitopes may begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab′)₂ fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries may be constructed(Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity.

[0108] Antibodies to the MC4-R can, in turn, be utilized to generateanti-idiotype antibodies that “mimic” the MC4-R, using techniques wellknown to those skilled in the art. (See, e.g., Greenspan & Bona, 1993,FASEB J 7(5):437-444; and Nissinoff, 1991, J. Immunol.147(8):2429-2438). For example antibodies which bind to the MC4-R ECDand competitively inhibit the binding of melanocortins to the MC4-R canbe used to generate anti-idiotypes that “mimic” the ECD and, therefore,bind and neutralize melanocortins. Such neutralizing anti-idiotypes orFab fragments of such anti-idiotypes can be used in therapeutic regimensto neutralize the native ligand and promote weight gain.

[0109] Alternatively, antibodies to MC4-R that can act as agonists ofMC4-R activity can be generated. Such antibodies will bind to the MC4-Rand activate the signal transducing activity of the receptor. Suchantibodies would be particularly useful for treating weight disorderssuch as obesity. In addition, antibodies that act as antagonist of MC4-Ractivity, i.e. inhibit the activation of MC4-R receptor may be used totreat weight disorders such as anorexia or cachexia.

[0110] 5.4. Gene Therapy Approaches to Controlling MC4-R Activity andRegulating Body Weight

[0111] The expression of MC4-R can be controlled in vivo (e.g. at thetranscriptional or translational level) using gene therapy approaches toregulate MC4-R activity and treat body weight disorders. Certainapproaches are described below.

[0112] 5.4.1. Gene Replacement Therapy

[0113] With respect to an increase in the level of normal MC4-R geneexpression and/or MC4-R gene product activity, MC4-R nucleic acidsequences can be utilized for the treatment of body weight disorders,including obesity. Where the cause of obesity is a defective MC4-R gene,treatment can be administered, for example, in the form of genereplacement therapy. Specifically, one or more copies of a normal MC4-Rgene or a portion of the MC4-R gene that directs the production of anMC4-R gene product exhibiting normal function, may be inserted into theappropriate cells within a patient or animal subject, using vectorswhich include, but are not limited to adenovirus, adeno-associatedvirus, retrovirus and herpes virus vectors, in addition to otherparticles that introduce DNA into cells, such as liposomes.

[0114] Because the MC4-R gene is expressed in the brain, including thecortex,-thalamus, brain stem and spinal cord and hypothalamus, such genereplacement therapy techniques should be capable of delivering MC4-Rgene sequences to these cell types within patients. Thus, the techniquesfor delivery of the MC4-R gene sequences should be designed to readilycross the blood-brain barrier, which are well known to those of skill inthe art (see, e.g., PCT application, publication No. WO89/10134, whichis incorporated herein by reference in its entirety), or, alternatively,should involve direct administration of such MC4-R gene sequences to thesite of the cells in which the MC4-R gene sequences are to be expressed.

[0115] Alternatively, targeted homologous recombination can be utilizedto correct the defective endogenous MC4-R gene in the appropriatetissue; e.g., brain tissue. In animals, targeted homologousrecombination can be used to correct the defect in ES cells in order togenerate offspring with a corrected trait.

[0116] Additional methods which may be utilized to increase the overalllevel of MC4-R gene-expression and/or MC4-R activity include theintroduction of appropriate MC4-R expressing cells, preferablyautologous cells, into a patient at positions and in numbers which aresufficient to ameliorate the symptoms of body weight disorders,including obesity. Such cells may be either recombinant ornon-recombinant. Among the cells which can be administered to increasethe overall level of MC4-R gene expression in a patient are normalcells, or hypothalamus cells which express the MC4-R gene. The cells canbe administered at the anatomical site in the brain, or as part of atissue graft located at a different site in the body. Such cell-basedgene therapy techniques are well known to those skilled in the art, see,e.g., Anderson, et al., U.S. Pat. No. 5,399,349; Mulligan & Wilson, U.S.Pat. No. 5,460,959.

[0117] Finally, compounds, identified in the assays described above,that stimulate or enhance the signal transduced by activated MC4-R,e.g., by activating downstream signalling proteins in the MC4-R cascadeand thereby by passing the defective MC4-R, can be used to achieveweight loss. The formulation and mode of administration will depend uponthe physico-chemical properties of the compound. The administrationshould include known techniques that allow for a crossing of theblood-brain barrier.

[0118] 5.4.2. Inhibition of MC4-R Expression

[0119] In an alternate embodiment, weight gain therapy can be designedto reduce the level of endogenous MC4-R gene expression, e.g., usingantisense or ribozyme approaches to inhibit or prevent translation ofMC4-R mRNA transcripts; triple helix approaches to inhibit transcriptionof the MC4-R gene; or targeted homologous recombination to inactivate or“knock out” the MC4-R gene or its endogenous promoter. Such gene therapymay be utilized for treatment of body weight disorders such as cachexiaand anorexia where the inhibition of MC4-R expression is designed to increase body weight. Because the MC4-R gene is expressed in the brain,delivery techniques should be preferably designed to cross theblood-brain barrier (see PCT WO89/10134, which is incorporated byreference herein in its entirety). Alternatively, the antisense,ribozyme or DNA constructs described herein could be administereddirectly to the site containing the target cells.

[0120] Antisense approaches involve the design of oligonucleotides(either DNA or RNA) that are complementary to mRNA. The antisenseoligonucleotides will bind to the complementary mRNA transcripts andprevent translation. Absolute complementarily, although preferred, isnot required. A sequence complementary to a portion of an RNA, asreferred to herein, means a sequence having sufficient complementarilyto be able to hybridize with the RNA, forming a stable duplex; in thecase of double-stranded antisense nucleic acids, a single strand of theduplex DNA may thus be tested, or triplex formation may be assayed. Theability to hybridize will depend on both the degree of complementarilyand the length of the antisense nucleic acid. Generally, the longer thehybridizing nucleic acid, the more base mismatches with an RNA it maycontain and still form a stable duplex (or triplex, as the case may be).One skilled in the art can ascertain a tolerable degree of mismatch byuse of standard procedures to determine the melting point of thehybridized complex.

[0121] While antisense nucleotides complementary to the coding regionsequence could be used, those complementary to the transcribeduntranslated region are most preferred. Oligonucleotides that arecomplementary to the 5′ end of the message, e.g., the 5′ untranslatedsequence up to and including the AUG initiation codon, should work mostefficiently at inhibiting translation (see FIG. 5). However, sequencescomplementary to the 3′ untranslated sequences of mRNAs have recentlyshown to be effective at inhibiting translation of mRNAs as well. Seegenerally, Wagner, R., 1994, Nature 372:333-335. Thus, oligonucleotidescomplementary to either the 5′- or 3′-non-translated, non-coding regionsof MC4-R could be used in an antisense approach to inhibit translationof endogenous mRNA. Oligonucleotides complementary to the 5′untranslated region of the mRNA should include the complement of the AUGstart codon. Antisense oligonucleotides complementary to mRNA codingregions are less efficient inhibitors of translation but could be usedin accordance with the invention. Whether designed to hybridize to the5′-, 3′- or coding region of MC4-R mRNA, antisense nucleic acids shouldbe at least six nucleotides in length, and are preferablyoligonucleotides ranging from 6 to about 50 nucleotides in length. Inspecific aspects the oligonucleotide is at least 10 nucleotides, atleast 17 nucleotides, at least 25 nucleotides or at least 50nucleotides.

[0122] Regardless of the choice of target sequence, it is preferred thatin vitro studies are first performed to quantitate the ability of theantisense oligonucleotide to inhibit gene expression. It is preferredthat these studies utilize controls that distinguish between antisensegene inhibition and nonspecific biological effects of oligonucleotides.It is also preferred that these studies compare levels of the target RNAor protein with that of an internal control RNA or protein.Additionally, it is envisioned that results obtained using the antisenseoligonucleotide are compared with those obtained using a controloligonucleotide. It is preferred that the control oligonucleotide is ofapproximately the same length as the test oligonucleotide and that thenucleotide sequence of the oligonucleotide differs from the antisensesequence no more than is necessary to prevent specific hybridization tothe target sequence.

[0123] The oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci.U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci.84:648-652; PCT Publication No. WO88/09810, published Dec. 15, 1988) orthe blood-brain barrier (see, e.g., PCT Publication No. WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalatingagents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

[0124] The antisense oligonucleotide may comprise at least one modifiedbase moiety which is selected from the group including but not limitedto 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic-acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[0125] The antisense oligonucleotide may also comprise at least onemodified sugar moiety selected from the group including but not limitedto arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0126] In yet another embodiment, the antisense oligonucleotidecomprises at least one modified phosphate backbone selected from thegroup consisting of a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

[0127] In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

[0128] Oligonucleotides of the invention may be synthesized by standardmethods known in the art, e.g. by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides may be synthesizedby the method of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc. The antisense molecules should be deliveredto cells which express the MC4-R in vivo, e.g., neural tissue. A numberof methods have been developed for delivering antisense DNA or RNA tocells; e.g., antisense molecules can be injected directly into thetissue site, or modified antisense molecules., designed to target thedesired cells (e.g., antisense linked to peptides or antibodies thatspecifically bind receptors or antigens expressed on the target cellsurface) can be administered systemically.

[0129] However, it is often difficult to achieve intracellularconcentrations of the antisense sufficient to suppress translation ofendogenous “RNA”. Therefore a preferred approach utilizes a recombinantDNA construct in which the antisense oligonucleotide is placed under thecontrol of a strong pol III or pol II promoter. The use of such aconstruct to transfect target cells in the patient will result in thetranscription of sufficient amounts of single stranded RNAs that willform complementary base pairs with the endogenous MC4-R transcripts andthereby prevent translation of the MC4-R mRNA. For example, a vector canbe introduced in vivo such that it is taken up by a cell and directs thetranscription of an antisense RNA. Such a vector can remain episomal orbecome chromosomally integrated, as long as it can be transcribed toproduce the desired antisense RNA. Such vectors can be constructed byrecombinant DNA technology methods standard in the art. Vectors can beplasmid, viral, or others known in the art, used for replication andexpression in mammalian cells. Expression of the sequence encoding theantisense RNA can be by any promoter known in the art to act inmammalian, preferably human cells. Such promoters can be inducible orconstitutive. Such promoters include but are not limited to: the SV40early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidinekinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.78:1441-1445), the regulatory sequences of the metallothionein gene(Brinster et al., 1982, Nature 296:39-42), etc. Any type of plasmid,cosmid, YAC or viral vector can be used to prepare the recombinant DNAconstruct which can be introduced directly into the tissue site; e.g.,the choroid plexus or hypothalamus. Alternatively, viral vectors can beused which selectively infect the desired tissue; (e.g., for brain,herpesvirus vectors may be used), in which case administration may beaccomplished by another route (e.g., systemically).

[0130] Ribozyme molecules designed to catalytically cleave MC4R mRNAtranscripts can also be used to prevent translation of MC4-R andexpression of MC4-R. (See, e.g., PCT International PublicationWO90/11364, published Oct. 4, 1990; Sarver et al., 1990, Science247:1222-1225). While ribozymes that cleave mRNA at site specificrecognition sequences can be used to destroy MC4-R mRNAs, the use ofhammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs atlocations dictated by flanking regions that form complementary basepairs with the target mRNA. The sole requirement is that the target mRNAhave the following sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art and isdescribed more fully in Haseloff and Gerlach, 1988, Nature, 334:585-591.There are hundreds of potential hammerhead ribozyme cleavage siteswithin the nucleotide sequence of human MC4-R cDNA (see FIG. 5).Preferably the ribozyme is engineered so that the cleavage recognitionsite is located near the 5′ end of the MC4-R mRNA; i.e., to increaseefficiency and minimize the intracellular accumulation of non-functionalmRNA transcripts.

[0131] The ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”) such as the onewhich occurs naturally in Tetrahymena Thermophila (known as the IVS, orL-19 IVS RNA) and which has been extensively described by Thomas Cechand collaborators (Zaug, et al., 1984, Science, 224:574-578; Zaug andCech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature,324:429-433; published International patent application No. WO 88/04300by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216). TheCech-type ribozymes have an eight base pair active site which hybridizesto a target RNA sequence whereafter cleavage of the target RNA takesplace. The invention encompasses those Cech-type ribozymes which targeteight base-pair active site sequences that are present in MC4-R.

[0132] As in the antisense approach, the ribozymes can be composed ofmodified oligonucleotides (e.g. for improved stability, targeting, etc.)and should be delivered to cells which express the MC4-R in vivo, e.g.,hypothalamus. A preferred method of delivery involves using a DNAconstruct “encoding” the ribozyme under the control of a strongconstitutive pol III or pol II promoter, so that transfected cells willproduce sufficient quantities of the ribozyme to destroy endogenousMC4-R messages and inhibit translation. Because ribozymes unlikeantisense molecules, are catalytic, a lower intracellular concentrationis required for efficiency.

[0133] Endogenous MC4-r gene expression can also be reduced byinactivating or “knocking out” the MC4-r gene or its promoter usingtargeted homologous recombination (e.g., see Smithies et al., 1985,Nature 317:230-234; Thomas & Capecchi, 1987, Cell 51:503-512; Thompsonet al., 1989 Cell 5:313-321; each of which is incorporated by referenceherein in its entirety). For example, a mutant, non-functional MC4-R (ora completely unrelated DNA sequence) flanked by DNA homologous to theendogenous MC4-r gene can be used, with or without a selectable markerand/or a negative selectable marker, to transfect cells that expressMC4-R in vivo. Insertion of the DNA construct, via targeted homologousrecombination, results in inactivation of the MC4-r gene. Suchapproaches are particularly suited in the agricultural field wheremodifications to ES (embryonic stem) cells can be used to generateanimal offspring with an inactive MC4-R (e.g., see Thomas & Capecchi1987 and Thompson 1989, supra). However this approach can be adapted foruse in humans provided the recombinant DNA constructs are directlyadministered or targeted to the required site in vivo using appropriateviral vectors, e.g., herpes virus vectors for delivery to brain tissue;e.g., the hypothalamus and/or choroid plexus.

[0134] Alternatively, endogenous MC4-R gene expression can be reduced bytargeting deoxyribonucleotide sequences complementary to the regulatoryregion of the MC4-R gene (i.e., the MC4-R promoter and/or enhancers) toform triple helical structures that prevent transcription of the MC4-Rgene in target cells in the body. (See generally, Helene, C. 1991,Anticancer Drug Des., 6(6):569-84; Helene, C., et al., 1992, Ann, N.Y.Acad. Sci., 660:27-36; and Maher, L. J., 1992, Bioassays 14(12):807-15).

[0135] 5.4.3. Delivery of Soluble MC4-R Polypeptides

[0136] Genetically engineered cells that express soluble MC4-R ECDs orfusion proteins e.g. fusion Ig molecules can be administered in vivowhere they may function as “bioreactors” that deliver a supply of thesoluble molecules. Such soluble MC4-R polypeptides and fusion proteins,when expressed at appropriate concentrations, should neutralize or “mopup” the native ligand for MC4-R, and thus act as inhibitors of MC4-Ractivity and induce weight gain.

[0137] 5.5. Pharmaceutical Formulations and Methods of Treating BodyWeight Disorders

[0138] The invention encompasses methods and compositions for modifyingbody weight and treating body weight disorders, including but notlimited to obesity, cachexia and anorexia. Because a loss of normalMC4-R gene product function results in the development of an obesephenotype, an increase in MC4-R gene product activity, or activation ofthe MC4-R pathway (e.g., downstream activation) would facilitateprogress towards a normal body weight state in obese individualsexhibiting a deficient level of MC4-R gene expression and/or MC4-Ractivity.

[0139] Alternatively, symptoms of certain body weight disorders such as,for example, cachexia, which involve a lower than normal body weightphenotype, may be ameliorated by decreasing the level of MC4-R geneexpression, and/or MC4-R gene activity, and/or downregulating activityof the MC4-R pathway (e.g., by targeting downstream signalling events).Different approaches are discussed below.

[0140] Agonists of MC4-R can be used to induce weight loss for treatingobesity. Antagonists of MC4-R activity can be used to induce weight gainfor treating conditions such as anorexia or cachexia. It is notnecessary that the compound demonstrate absolute specificity for theMC4-R. For example, compounds which agonize both MC4-R and MC1-R couldbe used; such compounds could be administered so that delivery to thebrain is optimized to achieve weight reduction, and side effects, suchas peripheral melanin production resulting in a “tan” may well betolerated. Compounds which do not demonstrate a specificity for MC4-Rcan be administered in conjunction with another therapy or drug tocontrol the side-effects that may result from modulating anothermelanocortin receptor; however, compounds which demonstrate a preferenceor selectivity for MC4-R over MC3-R are preferred since both receptorsare expressed in the brain where localized delivery cannot be used tocompensate for lack of receptor specificity.

[0141] 5.5.1. Dose Determinations

[0142] Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. Compounds which exhibit large therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

[0143] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED so with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

[0144] 5.5.2. Formulations and Use

[0145] Pharmaceutical compositions for use in accordance with thepresent invention may be formulated in conventional manner using one ormore physiologically acceptable carriers or excipients.

[0146] Thus, the compounds and their physiologically acceptable saltsand solvates may be formulated for administration by inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration. For oral administration, thepharmaceutical compositions may take the form of, for example, tabletsor capsules prepared by conventional means with pharmaceuticallyacceptable excipients such as binding agents (e.g., pregelatinised maizestarch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers(e.g., lactose, microcrystalline cellulose or calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc or silica);disintegrants (e.g., potato starch or sodium starch glycolate); orwetting agents (e.g., sodium lauryl sulphate). The tablets may be coatedby methods well known in the art. Liquid preparations for oraladministration may take the form of, for example, solutions, syrups orsuspensions, or they may be presented as a dry product for constitutionwith water or other suitable vehicle before use. Such liquidpreparations may be prepared by conventional means with pharmaceuticallyacceptable additives such as suspending agents (e.g., sorbitol syrup,cellulose derivatives or hydrogenated edible fats); emulsifying agents(e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil,oily-esters, ethyl alcohol or fractionated vegetable oils); andpreservatives (e.g., methyl- or propyl-p-hydroxybenzoates or sorbicacid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

[0147] Preparations for oral administration may be suitably formulatedto give controlled release of the active compound. For buccaladministration the compositions may take the form of tablets or lozengesformulated in conventional manner.

[0148] For administration by inhalation, the compounds for use accordingto the present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

[0149] The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

[0150] The compounds may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

[0151] In addition to the formulations described previously, thecompounds may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

[0152] The compositions may, if desired, be presented in a pack ordispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

6. EXAMPLE Generation of an MC4-R Deficient Mouse

[0153] The following example describes the engineering and generation of“knock-out” mice in which the endogenous MC4-r is inactivated. Theresults show that the knock-out mice gain weight, thus, demonstratingthe role and function of the MC4-R in body weight regulation.

[0154] 6.1. Materials and Methods

[0155] 6.1.1. Identification of the Murine MC4-R Gene

[0156] The murine melanocortin 4 receptor (MC4-r) gene was isolated froma mouse strain 129/Sv genomic phage library, obtained from Stratagene,using a human MC4-r probe. The human probe was generated by PCRamplification of MC4-r coding sequences from human genomic DNA using thefollowing primers:

[0157] 5′-ATA GTC GAC ATG GTG AAC TCC ACC CAC CGT-3′; and

[0158] 5′-TAT AAG CTT TTA ATA TCT GCT AGA CAA GTC-3′.

[0159] Two positive phage clones containing the MC4-r gene wereidentified, and the MC4-r locus was subcloned from phage intopBluescript II as an −5 Kb Hind III fragment, and an −4.7 Kb Sac Ifragment. These subclones were restriction mapped and partiallysequenced to produce the map of the MC4-r locus shown in FIG. 2A. Inorder to inactivate MC4-r, a targeting construct was built which woulddelete the majority of MC4-r coding sequences following homologousrecombination with the endogenous MC4-r locus.

[0160] 6.1.2. Generation of the Targeting Construct

[0161] The MC4-r targeting construct was constructed in the followingmanner. The 1.4 Kb Eco RI-Ava I fragment of pBR322 was replaced with thefollowing synthetic oligonucleotides:

[0162] 5′-AAT TAG CGG CCG CAG TAT GCA AAA AAA AGC CCG CTC ATT AGG CGGGCT-3′; and 5′-CCG AAG CCC GCC TAA TGA GCG GGC TTT TTT TTG CAT ACT GCGGCC GCT-3′.

[0163] The resulting plasmid, called pJN1, was digested with Not I andthe following oligonucleotides were ligated into the Not I site.

[0164] 5′-GGC CGG CAT GCA TCA AGC TTA TCT CGA GAT CGT CGA CTA CCA TGGTAC ATC GAT CAG GTA CCA TCC CGG GGC-3′; and 5′GGC CGC CCC GGG ATG GTACCT GAT CGA TGT ACC ATG GTA GTC GAC GAT CTC GAG ATA AGC TTG ATG CATGCC-3′.

[0165] The resulting plasmid was called pJN2.

[0166] The 1.2 Kb Sph I-Hind III fragment 3′of the MC4-r gene (see FIG.2A) was subcloned into SphI-Hind III digested pJN2 to generate theplasmid MC4-r KO 3′ (FIG. 2B). This fragment represents the 3′ region ofgenomic homology in the targeting vector. A 3.4 Kb NcoI-Hind IIIfragment, including the first approximately 20 nucleotides of the MC4-rgene (see FIG. 2A), was excised as a NcoI-Asp718 fragment from thesubclone MC4-r locus. The Asp718 site was derived from pBluescript IIpolylinker sequences immediately flanking the native Hind III siteapproximately 3.4 Kb 5′ of the MC4-r gene (FIG. 2A). This fragment,which represents the 5′ region of genomic homology in the targetingconstruct, was ligated into NcoI-Asp 718 digested MC4-r KO 5′ to produceMC4-r KO 5′3′ (FIG. 2C).

[0167] The PGK-neo expression cassette from the plasmid pKJ1 (Tybulewiczet al., Cell 65, 1153-1163, 1991), containing the neo gene under thetranscriptional control of the mouse phosphoglycerate kinase (PGK-1)promoter and the PGK-1 poly(A) addition site, was subcloned as an EcoRI-Hind III fragment into EcoRI-Hind III digested PGEM 7-Zf(+) togenerate PGEM 7 (KJ1). The 1.7 Kb fragment containing the PGK-neoexpression cassette was excised by: 1) digestion of pGEM 7 (KJ1) withXho I, which cuts in the polylinker 5′ of the PGK promoter, and bluntend filling of the Xho I site with Klenow polymerase, and 2) digestionwith Sca I which cuts within mouse genomic sequence 3′ of the PGKpolyadenylation signal. This fragment was ligated into Xho I digestedMC4-r KO 5′3′ which had also been blunt-ended with Klenow polymerase, togenerate the MC4-r targeting vector MC4-r KO 5131 neo (FIG. 2D). Aschematic map of the gene targeting strategy for inactivation of theMC4-r locus with this vector is shown in FIGS. 3A-3D.

[0168] 6.1.3. Generation of Targeted ES Cells

[0169] The RF-8 ES cell line (obtained from the Gladstone Institute ofCardiovascular Disease, UCSF) was cultured on SNL76/7 mitoticallyinactive feeder cells as described in McMahon and Bradley (1990 Cell 62:1073-1085). For electroporation, cells were trypsinized and resuspendedat a concentration of 1.1×10⁷/ml in PBS (Ca²+-and MG²+free; Gibco). An0.9 ml aliquot (1×10⁷ cells) was mixed with 20 μg of MC4-r KO 513′ DNA,which had been linearized by Not I digestion, and pulsed at 250V, 500 μF(Bio-Rad Gene Pulser), after which the cells were diluted in culturemedium, plated at 1×10⁶ per 100 mm plate containing feeder cells, andplaced under selection twenty-four hours later in G418 sulfate (400μg/ml powder, Gibco) for 6 days. 427 G418 resistant clones were picked,dissociated with trypsin and divided into one well each of two 96-wellplates. Upon confluence, ES cells were frozen in one of the 96-wellplates as described by Ramirez-Solis et al., (Methods in Enzymology,vol. 225, Wassarman, P. M., DePamphilis, M. L. (eds). Academic Press, p.855-878, 1992) and expanded into a 24 well plate. Upon confluence, DNAwas prepared for Southern blot analysis. Genomic DNA was prepared insitu from ES cells in 24 well plates by the procedure of Laird et al.(1991, Nucleic Acids Research 19: 4293). To screen for homologousrecombination between the vector and the endogenous MC4-r locusapproximately 20 μg of genomic DNA was digested with Apa I,electrophoresed through a 1% agarose gel, transferred to Hybond N⁺membrane (Amersham), and hybridized with the ³²P radiolabeled Sac I-SphI probe (see FIG. 3A). One of the 408 colonies screened showed theapproximately 7.6 Kb band expected of a gene targeting event, inaddition to the wild type 2.2 Kb band. This clone, number 155, wasfurther digested with Bam HI, Nco I, and Eco RI to verify thathomologous recombination had taken place between the targeting vectorand the MC4-r gene. In each case the expected bands of approximately2.1, 1.9, and 8.4 Kb, respectively, which are diagnostic of thepredicted gene targeting event, were observed. In addition to theexpected wild type bands of approximately 17 Kb, 2.8 Kb and 2.6 Kb, and9.9 Kb, respectively (see FIG. 3).

[0170] 6.1.4. Generation of MC4-R Deficient Mice

[0171] Clone 155 was injected into C57BL/6J blastocysts to generatechimeric mice as described in (Bradley, A. In Robertson, E. J. (ed)Teratocarcinomas and Embryonic Stem Cells. IRL Press, Oxford, England,p. 113-151, 1987). Male chimeras were bred with C57BL/6J females, andagouti offspring (representing germline transmission of the ES genome)were screened for the presence of the targeted MC4-r gene by Southernblot hybridization of Apa I as well as Kco I digested tail DNA using theprobe shown in FIG. 3A. Offspring heterozygous for the mutation wereidentified by either the presence of a 7.6 Kb Apa I band in addition tothe wild type 2.2 Kb band or the presence of a 1.9 Kb-Nco I band inaddition to the wild type bands of 2.6 Kb and 2.8 Kb (FIG. 3D).

[0172] Heterozygous mice were interbred and offspring generated by thesematings were screened by Southern blot hybridization of Apa I as well asNco I digested tail DNA. Mice homozygous for the deleted MC4-r gene wereidentified by the absence of the wild type 2.2 Kb Apa I band and 2.8 KbNco I band, and presence of the targeted 7.6 Kb Apa I band and 1.9 KbNco I band. To verify deletion of the MC4-r gene, the Apa I digested andNco I digested blots were stripped and re-probed with the human MC4-rcoding sequence. No hybridizing bands were observed in the DNA from micehomozygous for the MC4-r mutation, verifying the absence of the MC4-rgene in these mice.

[0173] 6.2. Results

[0174] 6.2.1. Weight Gain in MC4-R Deficient Mice

[0175] Weight gain of 7 females and 8 males homozygous for the MC4-rdeletion, 9 females and 10 males heterozygous for the MC4-r deletion,and 9 female and 10 male wild type littermates were monitored regularly.The weight gain of these mice is depicted in FIG. 4. Each linerepresents an individual mouse. The difference in lengths of the linesreflects the difference in ages of the various mice being tested. InFIG. 4A, the open arrows represent female mice homozygous for MC4-rdeletion, unmarked mice are wild type female littermates. FIG. 4Bdepicts the weight gain in male mice homozygous for MC4-r as indicatedby open arrows. Unmarked mice are wild type males. In FIG. 4C, theclosed arrows indicate female mice heterozygous for MC4-r deletion,unmarked mice are wild type female littermates. In FIG. 4D, the closedarrows indicate male mice heterozygous for MC4-r deletion, unmarked miceare wild type littermates.

[0176] Increased weight of homozygous, compared to heterozygous and wildtype mice, was observed as early as 25 days of age and is evident in allMC4-R deficient mice by approximately 50-60 days of age (FIG. 4). Theoldest homozygous mutant mice are two females which, at 94-98 days ofage, are on average over 2 fold heavier than their wild type littermatesand approximately 1.8 fold heavier than heterozygous littermates. Miceheterozygous for the MC4-r deletion tend, on average, to be slightlyheavier than wild type littermates, (FIG. 4C and D); an effect which ismore pronounced in female (FIG. 4C) compared to male mice (FIG. 4D). Thedata presented in FIG. 4 represents the first definitive evidence thatMC4-R plays an important role in weight regulation and indicates thatcompounds identified as regulators of MC4-R activity, i.e., agonist orantagonist of MC4-R, can be useful for treating various weightdisorders.

7. EXAMPLE Agouti Protein Binds Directly to MC1-R and MC4-R

[0177] The following example describes experiments demonstrating thatthe Agouti protein binds directly to the melanocortin receptors.

[0178] 7.1 Material and Methods

[0179] Human melanocortin receptor 4 (hMC4-r) cDNA, under the control ofthe CMV promoter, was transfected into the 293 cell line, and stableclones were selected (293/MC4-R). The stable clones were tested forreduction in intracellular cAMP levels in the presence of 5 nM agoutiprotein.

[0180] COS-7 cells were transfected with hMC1-r or hMC4-r by theDEAE-Dextran method. A plasmid containing the Adenovirus VA1 and VA2 RNAgenes was used to co-transfect the COS cells to enhance transientprotein expression by increasing translational initiation. The MC4-r andthe VA½ cDNA plasmids were used at a ratio of 10 to 1. Control platesreceived the VA½ plasmid alone. 48 hours post-transfection, the cellswere rinsed and culture supernatant containing 15 nm of AP-Ag was added.AP-Ag is a truncated agouti protein, containing the cysteine-rich domaintagged with the alkaline phosphatase at its N-terminal. Binding of AP-Agto transfected cells proceeded at room temperature for 90 minutes withgentle rocking. The cells were then washed 7 times before fixing andcolor development using NBT/BCIP substrate.

[0181] 7.2 Results

[0182] When 5 nM agouti protein was added to the 293/MC4-R line, an 18%reduction of intracellular cAMP level, relative to the parental 293 cellline, was observed. The results indicate that MC4-R mediates theagouti-triggered decrease of intracellular cAMP level and confers agoutiresponse to 293 cells.

[0183] N-terminal truncated agouti protein, containing only thecysteine-rich C-terminal domain, retains the antagonizing activity ofthe full-length agouti. A truncated agouti protein, containing only thecysteine-rich domain and tagged with the alkaline phosphatase at itsN-terminal, was used to assay the direct binding between the agoutiprotein and the MC1 and MC4 receptors in COS7 cells.

[0184] The transfection efficiency, as monitored by β-galactosidasereporter plasmid, typically was 12-16%. 10-13% of the MC1-r transfectedand 3-4% of the MC4-r transfected COS7 cells bound the agouti protein asdetermined by AP staining. The observed difference in percentage ofpositive cell between the MC1-r and MC4-r transfected COS7 cells couldbe attributed to differences in binding affinity and/or expressionlevel.

[0185] To assess the affinity of the agouti protein for the MC1-Rreceptor, a Scatchard Analysis was performed on MC1-r transfected COS7cells using culture supernatant containing up to 30 nM AP-Ag. The Kd hasbeen estimated at 20-30 nM range.

[0186] The present invention is not to be limited in scope by thespecific embodiments described which are intended as singleillustrations of individual apsects of the invention, and functionallyequivalent methods and components are within the scope of the invention.Indeed various modifications of the invention, in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and accompanying drawings. Suchmodifications are intended to fall within the scope of the appendedclaims.

1 12 1 360 PRT Homo sapiens 1 Met Ser Ile Gln Lys Lys Tyr Leu Glu GlyAsp Phe Val Phe Pro Val 1 5 10 15 Ser Ser Ser Ser Phe Leu Arg Thr LeuLeu Glu Pro Gln Leu Gly Ser 20 25 30 Ala Leu Leu Thr Ala Met Asn Ala SerCys Cys Leu Pro Ser Val Gln 35 40 45 Pro Thr Leu Pro Asn Gly Ser Glu HisLeu Gln Ala Pro Phe Phe Ser 50 55 60 Asn Gln Ser Ser Ser Ala Phe Cys GluGln Val Phe Ile Lys Pro Glu 65 70 75 80 Ile Phe Leu Ser Leu Gly Ile ValSer Leu Leu Glu Asn Ile Leu Val 85 90 95 Ile Leu Ala Val Val Arg Asn GlyAsn Leu His Ser Pro Met Tyr Phe 100 105 110 Phe Leu Cys Ser Leu Ala ValAla Asp Met Leu Val Ser Val Ser Asn 115 120 125 Ala Leu Glu Thr Ile MetIle Ala Ile Val His Ser Asp Tyr Leu Thr 130 135 140 Phe Glu Asp Gln PheIle Gln His Met Asp Asn Ile Phe Asp Ser Met 145 150 155 160 Ile Cys IleSer Leu Val Ala Ser Ile Cys Asn Leu Leu Ala Ile Ala 165 170 175 Val AspArg Tyr Val Thr Ile Phe Tyr Ala Leu Arg Tyr His Ser Ile 180 185 190 MetThr Val Arg Lys Ala Leu Thr Leu Ile Val Ala Ile Trp Val Cys 195 200 205Cys Gly Val Cys Gly Val Val Phe Ile Val Tyr Ser Glu Ser Lys Met 210 215220 Val Ile Val Cys Leu Ile Thr Met Phe Phe Ala Met Met Leu Leu Met 225230 235 240 Gly Thr Leu Tyr Val His Met Phe Leu Phe Ala Arg Leu His ValLys 245 250 255 Arg Ile Ala Ala Leu Pro Pro Ala Asp Gly Val Ala Pro GlnGln His 260 265 270 Ser Cys Met Lys Gly Ala Val Thr Ile Thr Ile Leu LeuGly Val Phe 275 280 285 Ile Phe Cys Trp Ala Pro Phe Phe Leu His Leu ValLeu Ile Ile Thr 290 295 300 Cys Pro Thr Asn Pro Tyr Cys Ile Cys Tyr ThrAla His Phe Asn Thr 305 310 315 320 Tyr Leu Val Leu Ile Met Cys Asn SerVal Ile Asp Pro Leu Ile Tyr 325 330 335 Ala Phe Arg Ser Leu Glu Leu ArgAsn Thr Phe Arg Glu Ile Leu Cys 340 345 350 Gly Cys Asn Gly Met Asn LeuGly 355 360 2 332 PRT Homo sapiens 2 Met Val Asn Ser Thr His Arg Gly MetHis Thr Ser Leu His Leu Trp 1 5 10 15 Asn Arg Ser Ser Tyr Arg Leu HisSer Asn Ala Ser Glu Ser Leu Gly 20 25 30 Lys Gly Tyr Ser Asp Gly Gly CysTyr Glu Gln Leu Phe Val Ser Pro 35 40 45 Glu Val Phe Val Thr Leu Gly ValIle Ser Leu Leu Glu Asn Ile Leu 50 55 60 Val Ile Val Ala Ile Ala Lys AsnLys Asn Leu His Ser Pro Met Tyr 65 70 75 80 Phe Phe Ile Cys Ser Leu AlaVal Ala Asp Met Leu Val Ser Val Ser 85 90 95 Asn Gly Ser Glu Thr Ile IleIle Thr Leu Leu Asn Ser Thr Asp Thr 100 105 110 Asp Ala Gln Ser Phe ThrVal Asn Ile Asp Asn Val Ile Asp Ser Val 115 120 125 Ile Cys Ser Ser LeuLeu Ala Ser Ile Cys Ser Leu Leu Ser Ile Ala 130 135 140 Val Asp Arg TyrPhe Thr Ile Phe Tyr Ala Leu Gln Tyr His Asn Ile 145 150 155 160 Met ThrVal Lys Arg Val Gly Ile Ser Ile Ser Cys Ile Trp Ala Ala 165 170 175 CysThr Val Ser Gly Ile Leu Phe Ile Ile Tyr Ser Asp Ser Ser Ala 180 185 190Val Ile Ile Cys Leu Ile Thr Met Phe Phe Thr Met Leu Ala Leu Met 195 200205 Ala Ser Leu Tyr Val His Met Phe Leu Met Ala Arg Leu His Ile Lys 210215 220 Arg Ile Ala Val Leu Pro Gly Thr Gly Ala Ile Arg Gln Gly Ala Asn225 230 235 240 Met Lys Gly Ala Ile Thr Leu Thr Ile Leu Ile Gly Val PheVal Val 245 250 255 Cys Trp Ala Pro Phe Phe Leu His Leu Ile Phe Tyr IleSer Cys Pro 260 265 270 Gln Asn Pro Tyr Cys Val Cys Phe Met Ser His PheAsn Leu Tyr Leu 275 280 285 Ile Leu Ile Met Cys Asn Ser Ile Ile Asp ProLeu Ile Tyr Ala Leu 290 295 300 Arg Ser Gln Glu Leu Arg Lys Thr Phe LysGlu Ile Ile Cys Cys Tyr 305 310 315 320 Pro Leu Gly Gly Leu Cys Asp LeuSer Ser Arg Tyr 325 330 3 297 PRT Homo sapiens 3 Met Lys His Ile Ile AsnSer Tyr Glu Asn Ile Asn Asn Thr Ala Arg 1 5 10 15 Asn Asn Ser Asp CysPro Arg Val Val Leu Pro Glu Glu Ile Phe Phe 20 25 30 Thr Ile Ser Ile ValGly Val Leu Glu Asn Leu Ile Val Leu Leu Ala 35 40 45 Val Phe Lys Asn LysAsn Leu Gln Ala Pro Met Tyr Phe Phe Ile Cys 50 55 60 Ser Leu Ala Ile SerAsp Met Leu Gly Ser Leu Tyr Lys Ile Leu Glu 65 70 75 80 Asn Ile Leu IleIle Leu Arg Asn Met Gly Tyr Leu Lys Pro Arg Gly 85 90 95 Ser Phe Glu ThrThr Ala Asp Asp Ile Ile Asp Ser Leu Phe Val Leu 100 105 110 Ser Leu LeuGly Ser Ile Phe Ser Leu Ser Val Ile Ala Ala Asp Arg 115 120 125 Tyr IleThr Ile Phe His Ala Leu Arg Tyr His Ser Ile Val Thr Met 130 135 140 ArgArg Thr Val Val Val Leu Thr Val Ile Trp Thr Phe Cys Thr Gly 145 150 155160 Thr Gly Ile Thr Met Val Ile Phe Ser His His Val Pro Thr Val Ile 165170 175 Thr Phe Thr Ser Leu Phe Pro Leu Met Leu Val Phe Ile Leu Cys Leu180 185 190 Tyr Val His Met Phe Leu Leu Ala Arg Ser His Thr Arg Lys IleSer 195 200 205 Thr Leu Pro Arg Ala Asn Met Lys Gly Ala Ile Thr Leu ThrIle Leu 210 215 220 Leu Gly Val Phe Ile Phe Cys Trp Ala Pro Phe Val LeuHis Val Leu 225 230 235 240 Leu Met Thr Phe Cys Pro Ser Asn Pro Tyr CysAla Cys Tyr Met Ser 245 250 255 Leu Phe Gln Val Asn Gly Met Leu Ile MetCys Asn Ala Val Ile Asp 260 265 270 Pro Phe Ile Tyr Ala Phe Arg Ser ProGlu Leu Arg Asp Ala Phe Lys 275 280 285 Lys Met Ile Phe Cys Ser Arg TyrTrp 290 295 4 317 PRT Homo sapiens 4 Met Ala Val Gln Gly Ser Gln Arg ArgLeu Leu Gly Ser Leu Asn Ser 1 5 10 15 Thr Pro Thr Ala Ile Pro Gln LeuGly Leu Ala Ala Asn Gln Thr Gly 20 25 30 Ala Arg Cys Leu Glu Val Ser IleSer Asp Gly Leu Phe Leu Ser Leu 35 40 45 Gly Leu Val Ser Leu Val Glu AsnAla Leu Val Val Ala Thr Ile Ala 50 55 60 Lys Asn Arg Asn Leu His Ser ProMet Tyr Cys Phe Ile Cys Cys Leu 65 70 75 80 Ala Leu Ser Asp Leu Leu ValSer Gly Thr Asn Val Leu Glu Thr Ala 85 90 95 Val Ile Leu Leu Leu Glu AlaGly Ala Leu Val Ala Arg Ala Ala Val 100 105 110 Leu Gln Gln Leu Asp AsnVal Ile Asp Val Ile Thr Cys Ser Ser Met 115 120 125 Leu Ser Ser Leu CysPhe Leu Gly Ala Ile Ala Val Asp Arg Tyr Ile 130 135 140 Ser Ile Phe TyrAla Leu Arg Tyr His Ser Ile Val Thr Leu Pro Arg 145 150 155 160 Ala ProArg Ala Val Ala Ala Ile Trp Val Ala Ser Val Val Phe Ser 165 170 175 ThrLeu Phe Ile Ala Tyr Tyr Asp His Val Ala Val Leu Leu Cys Leu 180 185 190Val Val Phe Phe Leu Ala Met Leu Val Leu Met Ala Val Leu Tyr Val 195 200205 His Met Leu Ala Arg Ala Cys Gln His Ala Gln Gly Ile Ala Arg Leu 210215 220 His Lys Arg Gln Arg Pro Val His Gln Gly Phe Gly Leu Lys Gly Ala225 230 235 240 Val Thr Leu Thr Ile Leu Leu Gly Ile Phe Phe Leu Cys TrpGly Pro 245 250 255 Phe Phe Leu His Leu Thr Leu Ile Val Leu Cys Pro GluHis Pro Thr 260 265 270 Cys Gly Cys Ile Phe Lys Asn Phe Asn Leu Phe LeuAla Leu Ile Ile 275 280 285 Cys Asn Ala Ile Ile Asp Pro Leu Ile Tyr AlaPhe His Ser Gln Glu 290 295 300 Leu Arg Arg Thr Leu Lys Glu Val Leu ThrCys Ser Trp 305 310 315 5 1671 DNA Homo sapiens 5 agcttccgag aggcagccgatgtgagcatg tgcgcacaga ttcgtctccc aatggcatgg 60 cagcttcaag gaaaattattttgaacagac ttgaatgcat aagattaaag ttaaagcaga 120 agtgagaaca agaaagcaaagagcagactc tttcaactga gaatgaatat tttgaagccc 180 aagattttaa agtgatgatgattagagtcg tacctaaaag agactaaaaa ctccatgtca 240 agctctggac ttgtgacatttactcacagc aggcatggca attttagcct cacaactttc 300 agacagataa agacttggaggaaataactg agacgactcc ctgacccagg aggttaaatc 360 aattcagggg gacactggaattctcctgcc agcatggtga actccaccca ccgtgggatg 420 cacacttctc tgcacctctggaaccgcagc agttacagac tgcacagcaa tgccagtgag 480 tcccttggaa aaggctactctgatggaggg tgctacgagc aactttttgt ctctcctgag 540 gtgtttgtga ctctgggtgtcatcagcttg ttggagaata tcttagtgat tgtggcaata 600 gccaagaaca agaatctgcattcacccatg tactttttca tctgcagctt ggctgtggct 660 gatatgctgg tgagcgtttcaaatggatca gaaaccatta tcatcaccct attaaacagt 720 acagatacgg atgcacagagtttcacagtg aatattgata atgtcattga ctcggtgatc 780 tgtagctcct tgcttgcatccatttgcagc ctgctttcaa ttgcagtgga caggtacttt 840 actatcttct atgctctccagtaccataac attatgacag ttaagcgggt tgggatcagc 900 ataagttgta tctgggcagcttgcacggtt tcaggcattt tgttcatcat ttactcagat 960 agtagtgctg tcatcatctgcctcatcacc atgttcttca ccatgctggc tctcatggct 1020 tctctctatg tccacatgttcctgatggcc aggcttcaca ttaagaggat tgctgtcctc 1080 cccggcactg gtgccatccgccaaggtgcc aatatgaagg gagcgattac cttgaccatc 1140 ctgattggcg tctttgttgtctgctgggcc ccattcttcc tccacttaat attctacatc 1200 tcttgtcctc agaatccatattgtgtgtgc ttcatgtctc actttaactt gtatctcata 1260 ctgatcatgt gtaattcaatcatcgatcct ctgatttatg cactccggag tcaagaactg 1320 aggaaaacct tcaaagagatcatctgttgc tatcccctgg gaggcctttg tgacttgtct 1380 agcagatatt aaatggggacagagcacgca atataggaac atgcataaga gactttttca 1440 ctcttaccct acctgaatattgtacttctg caacagcttt ctcttccgtg tagggtactg 1500 gttgagatat ccattgtgtaaatttaagcc tatgattttt aatgagaaaa aatgcccagt 1560 ctctgtatta tttccaatgtcatgctactt ttttggccat aaaatatgaa tctatgttat 1620 aggttgtagg cactgtggatttacaaaaag aaaagtcctt attaaaagct t 1671 6 332 PRT Rattus sp. 6 Met ValAsn Ser Thr His Arg Gly Met His Thr Ser Leu His Leu Trp 1 5 10 15 AsnArg Ser Ser Tyr Arg Leu His Ser Asn Ala Ser Glu Ser Leu Gly 20 25 30 LysGly Tyr Ser Asp Gly Gly Cys Tyr Glu Gln Leu Phe Val Ser Pro 35 40 45 GluVal Phe Val Thr Leu Gly Val Ile Ser Leu Leu Glu Asn Ile Leu 50 55 60 ValIle Val Ala Ile Ala Lys Asn Lys Asn Leu His Ser Pro Met Tyr 65 70 75 80Phe Phe Ile Cys Ser Leu Ala Val Ala Asp Met Leu Val Ser Val Ser 85 90 95Asn Gly Ser Val Ser Ile Val Ile Thr Leu Leu Asn Ser Thr Asp Thr 100 105110 Asp Ala Gln Ser Phe Thr Val Asn Ile Asp Asn Val Ile Asp Ser Val 115120 125 Ile Cys Ser Ser Leu Leu Ala Ser Ile Cys Ser Leu Leu Ser Ile Ala130 135 140 Val Asp Arg Tyr Phe Thr Ile Phe Tyr Ala Leu Gln Tyr His AsnIle 145 150 155 160 Met Thr Val Arg Arg Val Gly Ile Ile Ile Ser Cys IleTrp Ala Ala 165 170 175 Cys Thr Val Ser Gly Val Leu Phe Ile Ile Tyr SerAsp Ser Ser Ala 180 185 190 Val Ile Ile Cys Leu Ile Thr Met Phe Phe ThrMet Leu Val Leu Met 195 200 205 Ala Ser Leu Tyr Val His Met Phe Leu MetAla Arg Leu His Ile Lys 210 215 220 Arg Ile Ala Val Leu Pro Gly Thr GlyAla Ile Arg Gln Gly Ala Asn 225 230 235 240 Met Lys Gly Thr Ile Thr LeuThr Ile Leu Ile Gly Val Phe Val Val 245 250 255 Cys Trp Ala Pro Phe PheLeu His Leu Leu Phe Tyr Ile Ser Cys Pro 260 265 270 Gln Asn Pro Tyr CysVal Cys Phe Met Ser His Phe Asn Leu Tyr Leu 275 280 285 Ile Leu Ile MetCys Asn Ser Ile Ile Asp Pro Leu Ile Tyr Ala Leu 290 295 300 Arg Ser GlnGlu Leu Arg Lys Thr Phe Lys Glu Ile Ile Cys Cys Tyr 305 310 315 320 ProLeu Gly Gly Leu Cys Asp Leu Ser Ser Arg Tyr 325 330 7 30 DNA ArtificialSequence Primer 7 atagtcgaca tggtgaactc cacccaccgt 30 8 30 DNAArtificial Sequence Primer 8 tataagcttt tatattctgc tagacaagtc 30 9 48DNA Artificial Sequence oligonucleotide 9 aattagcggc cgcagtatgcaaaaaaaagc ccgctcatta ggcgggct 48 10 48 DNA Artificial SequenceOligonucleotide 10 ccgaagcccg cctaatgagc gggctttttt ttgcatactg cggccgct48 11 72 DNA Artificial Sequence Oligonucleotide 11 ggccggcatgcatcaagctt atctcgagat cgtcgactac catggtacat cgatcaggta 60 ccatcccggg gc72 12 72 DNA Oligonucleotide 12 ggccgccccg ggatggtacc tgatcgatgtaccatggtag tcgacgatct cgagataagc 60 ttgatgcatg cc 72

What is claimed is:
 1. A method for the treatment of a body weightdisorder, comprising administering to an individual in need of treatmentan effective amount of a compound that agonizes or antagonizes theactivity of the melanocortin 4-receptor, so that the body weightdisorder is treated.
 2. The method of claim 1 in which the compound isan agonist that activates the melanocortin 4-receptor and induces weightloss.
 3. The method of claim 2 in which the compound is an antibody thatbinds to and activates the melanocortin 4-receptor.
 4. The method ofclaim 1 in which the compound is an antagonist that inhibits activationof the melanocortin 4-receptor and induces weight gain.
 5. The method ofclaim 4 in which the compound is an antibody or a soluble peptidecomprising an extracellular domain of the melanocortin 4-receptor.
 6. Amethod for the treatment of a body weight disorder, comprisingadministering to an individual in need of treatment an effective amountof a compound that modulates the activity of the melanocortin 4-receptorgene, so that the body weight disorder is treated.
 7. The method ofclaim 6, wherein the method comprises administering to an individual inneed of treatment an effective amount of a compound that decreasesexpression of the melanocortin 4-receptor and induces weight gain, sothat the body weight disorder is treated.
 8. The method of claim 7 inwhich the compound is an oligonucleotide comprising an antisense orribozyme molecule that targets melanocortin 4-receptor transcripts andinhibits translation.
 9. The method of claim 8 in which the compound isan oligonucleotide that forms a triple helix with the promoter of themelanocortin 4-receptor gene and inhibits transcription.